Single-stranded oligonucleotide probes for chromosome or gene copy enumeration

ABSTRACT

Single-stranded oligonucleotide probes, systems, kits and methods for chromosome enumeration, gene copy enumeration, or tissue diagnostics. The probes are particularly suited for detecting gene amplification, deletion, or rearrangement in tissue samples in a single, dual, or multiplexed assay. The probes exhibit improved performance compared to industry leading dual-stranded probes; particularly in terms of the rate of hybridization and the ability to achieve specific hybridization without blocking DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2015/068041 filed Aug. 5, 2015, which claims priority to andthe benefit of U.S. Provisional Patent Application No. 62/034,035, filedAug. 6, 2014. Each of the above patent applications is incorporatedherein by reference as if set forth in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 6, 2017, isnamed 32190_Project1_ST25.txt and is 1,291,542 bytes in size.

FIELD

This disclosure relates to oligonucleotide probes, systems, kits, andmethods for using said probes and systems for chromosome enumeration,for detection of nucleic acid target sequences (e.g., genomic DNA orRNA), for gene copy number enumeration, and/or for tissue diagnostics.

BACKGROUND

Probes have been developed for a variety of diagnostic and researchpurposes. Hybridization of chromosome or gene-specific probes has madepossible detection of chromosomal abnormalities associated with numerousdiseases and syndromes, including constitutive genetic anomalies, suchas microdeletion syndromes, chromosome translocations, geneamplification and aneuploidy syndromes, neoplastic diseases, as well aspathogen infections. Detection of genetic changes in these regions canprovide diagnostic and prognostic information for patients and in somecases, inform treatment decisions.

However, existing probes for detection and enumeration of humanchromosome 3 (CHR3) require long assay times and are not desirable. Forexample, chromosome 3 probes are typically generated from plasmid pHS05,which targets chromosome 3 alpha satellite sequences, or from BACs for asite-specific region (Yurov, Mitkevich et al. 1987; Woenckhaus, Stegeret al. 2007; Agell, Hernandez et al. 2011; Jang, Yonescu et al. 2006; DeMarco, Rinaldo et al. 2013; Nakayama et al. 2006). Unfortunately, thesedouble-stranded probes have repetitive sequences that are common tocentromere regions of other human chromosome. As such, blocking DNA isused in conjunction with these probes to help reduce non-specificbinding. Further, assays employing these probes require extensivehybridization time to achieve sufficient hybridization, e.g., about 12to 18 hours.

The use of single strand probes has a distinct advantage over the use ofdouble probes. Single-stranded probes generally have higher sensitivitythan double-stranded probes because a proportion of the denatureddouble-stranded probe renatures to form probe homoduplexes, thuspreventing their capture of genomic targets in the test samples [TanejaK and Singer R H. (1987), Lewis M E, Sherman T G, Watson S J. (1985);Strachan T, Read A P. (1999); Kourilsky P, Mercereau O, Gros D, TremblayG. (1974)]. Several laboratories have reported that single-strandedprobes provide higher sensitivity on hybridization than double-strandedprobes (An S F, Franklin D, Fleming K A. (1992); Hannon K, Johnstone E,Craft L S, et al (1993); Cox K H, DeLeon D V, Angerer L M, Angerer R C.(1984).

Despite the appeal of the use of a single strand probe in the detectionand enumeration of human chromosome 3, the general consensus by workersin this field is that it is not possible to make single strand probesthat are specific enough to chromosome 3. The major concern is theoff-target hits of a short single strand probe to other chromosometargets. Another concern is the robustness of a small number of shortsingle strand probes. It was found that human chromosome 3-specificalpha satellite contained a ˜2,900 base pair repeat unit that consistsof 17 monomers (Alexandrov, I. A., et al. (1993). Each individual maycarry different combinations of these 17 monomers and their relatedvariants. A limited number of short single strand probes (e.g., 2-5) maynot be robust enough to detect chromosome 3 polymorphism in populations(Yurov, Y. B., et al. 1987). As such, prior to the present invention,probes for detection and enumeration of human chromosome 3 are notdesirable and not time efficient.

Lung cancer is the leading cause of cancer-related mortality worldwide,with an estimate of 1.4 million deaths in 2010. In the United States,more than 220,000 new cases and more than 157,000 deaths annually withlung cancer, non-small cell lung cancer (NSCLC) accounts for 85% of alllung cancers. Adenocarcinoma and squamous-cell carcinoma (SCC) are themost frequent histological subtypes accounting for 50% and 30% of NSCLCcases respectively (Minna, Roth et al. 2002). Historically, approachesto the treatment of NSCLC were uniform, and histologic subtypes withinNSCLC did not significantly affect treatment decisions. In the past tenyears, knowledge of genetic aberrances of lung adenocarcinoma has beenidentified in most cases of lung adenocarcinoma; targeted drugs likeEGFR tyrosine kinase inhibitors and ALK inhibitors are now applied toabout one third of the patients (Zochbauer-Muller, Gazdar et al. 2002,Cooper, Lam et al. 2013, Villaruz, Burns et al. 2013). Unlikeadenocarcinoma of the lung, SCC remains a relatively poorly understooddisease that lacks a consensus molecular profile, although recent workis overcoming these challenges (Ji, Guan et al. 2011, Lockwood, Wilsonet al. 2012). Gain of chromosome 3q25-qter was revealed almostexclusively in SCC, among them the PIK3CA which encodes the catalyticalpha subunit (p110α) of phosphatidylinositol (PI) 3-kinase (PI3K) (Kok,Geering et al. 2009, Massion, Kuo et al. 2002, Okudela, Suzuki et al.2007). Moreover, PIK3CA was found to be amplified in up to 45% of SCCcancer patients (Yamamoto, Shigematsu et al. 2008, Spoerke, O'Brien etal. 2012). A strong correlation was found between PIK3CA amplificationand the increased PI3K pathway activity such as cell proliferation,survival, oncogenic RAS signaling and transformation (Woenckhaus, Stegeret al. 2002). These features make PI3K an attractive target fortherapeutic intervention on SCC of the lung (Wee, Lengauer et al. 2008).

Currently, several inhibitors directed against PI3K are being clinicallyevaluated for NSCLC treatment, where distinct candidate predictivebiomarkers strategies might be needed for SCC patient populations(Salphati, Belvin et al. 2009, Blumenthal, Orbach et al. 2011, Salphati,Pang et al. 2011). In a recent study by Spoerke J M et al. (Spoerke,O'Brien et al. 2012), PIK3CA amplification was detected with fluorescentin situ hybridization (FISH) in 37% of SCC and only 5% ofadenocarcinomas. Cell lines harboring PI3K amplification wereexquisitely sensitive to the PI3K inhibitor GDC-0941. In addition,Angulo B et al. (Angulo, Suarez-Gauthier et al. 2008) demonstrated astrong statistically significant association between increased levels ofPIK3CA mRNA expression and gene amplification predominantly in SCC. Onthe protein level, Scrima M et al. (Scrima, De Marco et al. 2012) showedthe majority of NSCLCs with increased copy number of PIK3CA showedmoderate or high expression of p110α.

Currently, all studies on PIK3CA copy number gain employ fluorescent insitu hybridization (FISH) technology. These PIK3CA FISH probes arederived from bacterial artificial chromosomes (BACs) that cover PIK3CAgene region; while most CHR3 probes are generated from plasmid pHS05that targets chromosome 3 alpha satellite sequences or BACs forsite-specific region. These double stranded probes require 12 to 18hours hybridization in order to achieve sufficient hybridization; andrequire blocking DNA due to the presence of repetitive sequence commonto other centromere regions which would result in non-specific bindingwithout the addition of the blocking DNA.

SUMMARY

The present inventors have surprisingly discovered a set of 18 uniquesingle strand probes that are highly specific for chromosome 3. In fact,these newly discovered oligonucleotide probes are so highly specificthat the use of blocking DNA can be eliminated in the assays.Furthermore, it was surprisingly discovered that these oligonucleotideprobes have much enhanced hybridization kinetics that they require asignificantly reduced hybridization time.

Also surprisingly, these single-stranded oligonucleotide probes tochromosome 3 resulted in discrete enumerable rounded signals. This is incontrast to the nick-translation labeled double-stranded probe, whichgenerate signals with a wide range of size and shape.

The single-stranded oligonucleotide probes to chromosome 3 of thepresent invention may be used in combination with one or more targetprobes directed to a target gene of interest. This allows for gene copyenumeration (e.g., determination of the ratio of a target gene to itscorresponding chromosome), which may be important for tissuediagnostics. As an example, PIK3CA is a gene found on chromosome 3.Commercial products and research reagents for PIK3CA gene copy numberuse bacterial artificial chromosomes (BACs) that cover the PIK3CA generegion (Angulo, Suarez-Gauthier et al. 2008); Shayesteh, Lu et al, 1999;Psyrri, Papageorgiou et al 2009). The BACs are double-stranded DNAprobes, which require about 12 to 18 hours hybridization time.

The present invention also features the use of single-strandedoligonucleotide probes to detect (and enumerate gene copy number) thePIK3CA gene on chromosome 3 in combination with chromosome 3 detectionand enumeration using the aforementioned single-stranded oligonucleotideprobes. This is the first and only demonstration of gene copy numberenumeration using single-stranded oligonucleotide probes for both thegene and chromosome targets.

In illustrative embodiments, systems for in situ hybridization maycomprise a control probe specific to a control region of a chromosome,e.g., chromosome 3. The control probe is configured to hybridize toformalin fixed paraffin embedded (FFPE) tissue in about 3 hours or less,e.g., 1 hour or less. In some embodiments, the control probe is aplurality of synthetic single-stranded oligonucleotides. The system mayalso feature a target probe specific to a target region of thechromosome, wherein the target probe is also configured to hybridize inabout a about 3 hours or less, e.g., 1 hour or less. In someembodiments, the control region is a centromere. The target region maybe a gene or gene locus.

In illustrative embodiments, systems for in situ hybridization maycomprise a control probe specific to a control region of chromosome 3,the control probe is labeled with at least one first label, the controlprobe is configured to achieve a staining intensity of ≧2 and stainingcoverage of ≧50% of the number of total nuclei of a control samplewithin 3 hours of hybridization.

In some embodiments, the systems may feature a target probe specific toa target region of chromosome 3, the target probe is labeled with atleast one label, the target probe is configured to achieve a stainingintensity of ≧2 and staining coverage of ≧50% of the number of totalnuclei of a target sample within 3 hours of hybridization.

In other illustrative embodiments, methods for in situ hybridization ofa tissue sample may comprise contacting the tissue sample with a controlprobe, hybridizing the control probe to the control region underconditions for a period of time less than about 3 hours, rinsing thesample to remove unbound probe, and detecting presence of the hybridizedprobe. The control may comprise a plurality of single-stranded labeledsynthetic oligonucleotides. In one embodiment, the method furthercomprises applying chromogenic detection reagents that recognize labelsand amplify the signal associated with the probes. In furtherembodiments, methods using and kits pertaining to the aforementionedsystems are disclosed.

In some illustrative embodiments, methods for obtaining two bright-fieldchromogenic in situ hybridization signals per cell may comprisecontacting a tissue sample containing a plurality of cells with acontrol probe specific to a control region of a single chromosome, theprobe selected so as to not evidently bind non-specifically in theabsence of blocking DNA; hybridizing the control probe to the controlregion of said chromosome; rinsing the sample to remove unbound probe;and detecting the presence of the hybridized probe via a chromogenicreagent so as to generate two bright-field chromogenic in situhybridization signals per cell.

Additional features of the present disclosure will become apparent tothose skilled in the art upon consideration of the following detaileddescription of illustrative embodiments exemplifying the best mode ofcarrying out the disclosure as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic view of the 3q26.32 region of chromosome 3. ThePIK3CA oligo probe covers the region between 178,922,283 and179,682,019.

FIG. 2 shows PIK3CA and CHR3 DISH staining on metaphase spreadchromosomes.

FIG. 3 shows PIK3CA and CHR3 DISH staining on Calu 3 cells.

FIG. 4 is a hybridization time course study.

FIG. 5 shows cross-reactive signals in a normal lung tissue stained withpHS05 plasmid (for chromosome 3) in the absence of human blocking DNA.

FIG. 6A is a photograph showing CHR3 staining with the pHS05 plasmidprobe.

FIG. 6B is an additional photograph showing CHR3 staining with the pHS05plasmid probe.

FIG. 6C is a photograph showing CHR3 staining with the probes of thepresent invention or with the pHS05 plasmid. Staining with probes of thepresent invention offers discrete CHR3 signals with generally uniformshape and size.

FIG. 6D is an additional photograph showing CHR3 staining with theprobes of the present invention or with the pHS05 plasmid. Staining withprobes of the present invention offers discrete CHR3 signals withgenerally uniform shape and size.

FIG. 7 shows that the PIK3CA/CHR3 ratio from the scores of 50 nuclei wasconsistent with the scores from the entire 100 nuclei enumeration whenchoosing 50 nuclei with the highest counts.

FIG. 8A is a graph illustrating the PIK3CA/CHR3 ratio profile on thetested tissue types of the 102 lung specimens.

FIG. 8B is a graph illustrating the average PIK3CA copy number profileon the tested tissue types of the 102 lung specimens.

FIG. 8C is a graph illustrating the average CHR3 copy number profiles onthe tested tissue types of the 102 lung specimens.

FIG. 9 shows cases with PIK3CA copy number gain evaluated by PIK3CA/CHR3ratio (>2) and average PIK3CA copy number (>4) per nuclei.

FIG. 10A illustrates p110α IHC on Calu-3 xenograft staining results onthe 102 lung tissues.

FIG. 10B illustrates p110α IHC staining results on the 102 lung tissues.

FIG. 10C illustrates PIK3CA mRNA ISH (C) staining results on the 102lung tissues.

FIG. 11A illustrates (i) PIK3CA/CHR3 DISH, (ii) PIK3CA mRNA ISH and(iii) p100α IHC staining on a SCC (F2) with gene copy gain, increasedmRNA and protein expression levels.

FIG. 11B shows (i) PIK3CA/CHR3 DISH, (ii) PIK3CA mRNA ISH and (iii)p100α IHC staining on a SCC (F3). PIK3CA gene copy number is 2.16 withratio 0.98, mRNA level is slightly elevated (H score 75), proteinexpression is normal (IHC intensity 1).

FIG. 11C shows (i) PIK3CA/CHR3 DISH, (ii) PIK3CA mRNA ISH and (iii)p100α IHC staining on a SCC (F5). PIK3CA gene copy number is 2.58 withratio 1.04, mRNA level is within normal range (H score 65), but proteinis overexpressed (IHC intensity 3, 50%).

FIG. 12 shows the distribution of the CHR3 signal radii and thestatistical analysis of the data.

FIG. 13 shows the signals used for measurements (radii) in FIG. 12.

FIG. 14A shows examples of concentric circles (top) and simple closedcurves (bottom) used for evaluating enumerable signals.

FIG. 14B shows additional examples of concentric circles (top) andsimple closed curves (bottom) used for evaluating enumerable signals.

SEQUENCES

The nucleic acid sequences provided herein are shown using standardletter abbreviations for nucleotide bases, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the provided sequences:

SEQ ID NOs: 1-18 are nucleic acid sequences of probes to humanchromosome 3.

SEQ ID NOs: 19-1230 are nucleic acid sequences of probes to the humanPIK3CA gene locus or nearby regions.

DETAILED DESCRIPTION

The present inventors are not aware of any successful work thus far byanyone to improve the reliability and conditions for detection andenumeration of human chromosome 3 using pHS05 plasmid-derived chromosome3 probe. In the inventors' hands, extensive studies have been performedon the pHS05 plasmid-derived chromosome 3 probe, trying to optimizetarget retrieval steps, stringency wash step, and chromogen developmentsteps, but they were not able to achieve improved quality andreliability for these assays. Additionally, the present inventors havebeen unable to develop assay steps to reduce or eliminate therequirement to use blocking DNA in such assays. FIG. 5 shows theexcessive background signals in a hybridization assay using the pHS05plasmid CHR3 probe without the use of blocking DNA.

Additionally, overwhelming evidence exists that single stranded probescannot be developed for the detection and enumeration of humanchromosome 3. For example, as shown in Table 1, bioinformatics researchrevealed that the 80nt sequences derived from the pHS05plasmid/chromosome 3 probe (the sequences of the centromere region ofchromosome 3) had high homology to several other chromosomes (e.g.,chromosome 5, 10, 12, 14, 16, 21, 22 etc.).

TABLE 1 SEQ ID NO SCORE START END QSIZE IDENTITY CHRO STRAND START ENDSPAN 1 80 1 80 80 100.00% 3 + 91978690 91978769 80 1 80 1 80 80 100.00%3 + 91918985 91919064 80 1 80 1 80 80 100.00% 3 + 91906555 91906634 80 180 1 80 80 100.00% 3 + 91883680 91883759 80 1 80 1 80 80 100.00% 3 +91855796 91855875 80 1 80 1 80 80 100.00% 3 + 91830105 91830184 80 1 801 80 80 100.00% 3 + 91798300 91798379 80 1 80 1 80 80 100.00% 3 +91749140 91749219 80 1 80 1 80 80 100.00% 3 + 91743696 91743775 80 1 801 80 80 100.00% 3 + 91729235 91729314 80 1 80 1 80 80 100.00% 3 +91688231 91688310 80 1 80 1 80 80 100.00% 3 + 91685340 91685419 80 1 801 80 80 100.00% 3 + 91659655 91659734 80 1 80 1 80 80 100.00% 3 +91623591 91623670 80 1 80 1 80 80 100.00% 3 + 91606580 91606659 80 1 801 80 80 100.00% 3 + 91572395 91572474 80 1 78 1 80 80 98.80% 3 −93712111 93712190 80 1 78 1 80 80 98.80% 3 − 93709226 93709305 80 1 78 180 80 98.80% 3 − 93706338 93706417 80 1 78 1 80 80 98.80% 3 − 9154932791549406 80 1 76 1 80 80 97.50% 3 − 91543887 91543966 80 1 70 1 80 8093.80% 3 − 91546102 91546181 80 1 70 1 80 80 93.80% 3 − 9154831191548390 80 1 70 1 80 80 93.80% 3 − 91552216 91552295 80 1 67 1 80 8092.50% 12 − 37253189 37266589 13401 1 64 1 80 80 90.00% Un_KI270317v1 −37199 37278 80 1 64 1 80 80 90.00% 5 − 49631520 49631599 80 1 64 1 80 8090.00% 12 − 34715481 34715560 80 1 63 1 80 80 88.40% 3 − 9370735693707434 79 1 63 1 80 80 90.00% 3 − 93710239 93715739 5501 1 63 1 80 8090.00% 12 − 37251774 37254004 2231 1 62 1 80 80 88.80% 5 − 4962345049623529 80 1 62 1 80 80 88.80% 5 − 49625489 49625568 80 1 62 1 80 8088.80% 5 − 49630841 49630920 80 1 62 1 80 80 88.80% 5 − 4964412149644200 80 1 62 1 80 80 88.80% 5 − 49617016 49617095 80 1 62 1 80 8088.80% 5 − 49614976 49615055 80 1 62 1 80 80 88.80% 5 − 4961016549610244 80 1 62 1 80 80 88.80% 16 − 38267076 38267155 80 1 62 1 80 8088.80% 16 − 38275894 38275973 80 1 62 1 80 80 88.80% 12 − 3471581634715895 80 1 61 1 79 80 88.70% 12 − 37260072 37260150 79 1 60 1 80 8091.70% Un_KI270411v1 − 383 2496 2114 1 60 1 80 80 87.50% Un_KI270304v1 −726 805 80 1 60 1 80 80 87.50% Un_KI270304v1 − 1406 1485 80 1 60 1 80 8087.50% 5 − 49615656 49615735 80 1 60 1 80 80 87.50% 5 − 4962209549622174 80 1 60 1 80 80 87.50% 3 − 91545424 91545503 80 1 60 1 80 8087.50% 3 − 91551539 91551618 80 1 60 1 80 80 91.70% 12 − 3483398634834121 136 1 60 1 80 80 87.50% 10 − 41839614 41839693 80 1 60 1 80 8087.50% 10 − 41841816 41841895 80 1 58 1 80 80 86.30% Un_KI270411v1 −1060 1139 80 1 58 4 75 80 87.40% 3 − 93706685 93706755 71 1 58 1 80 8086.30% 16 − 38267416 38267495 80 1 57 6 80 80 88.00% 3 − 9154236191542435 75 1 57 8 80 80 89.10% 12 − 34835059 34835131 73 1 56 1 80 8085.00% 12 − 34833646 34833725 80 1 56 5 80 80 86.90% 1 − 122462723122462798 76 1 54 9 80 80 87.50% 12 − 37237861 37237932 72 1 54 1 80 8083.80% 12 − 34821901 34821980 80 1 54 1 80 80 86.80% 10 − 4184147741841555 79 1 53 3 80 80 84.70% 4 − 67399102 67400538 1437 1 53 6 80 8085.40% 3 − 91540504 91540578 75 1 52 1 70 80 87.20% 12 − 3483331634833385 70 1 49 1 80 80 86.90% 10 − 41842156 41842234 79 1 49 7 80 8094.60% 1 − 121612371 121859704 247334 1 48 19 80 80 88.80% 12 − 3723650137236562 62 1 46 19 80 80 87.10% 12 − 34831051 34831112 62 1 45 29 80 8094.30% 20 − 28760701 28760922 222 1 44 23 80 80 88.00% Un_KI270366v1 −3039 3096 58 1 44 7 66 80 86.70% 9 − 63075458 63075517 60 1 44 7 80 8079.80% 10 − 41835720 41835793 74 1 43 29 80 80 92.20%5_GL000208v1_random − 44114 44477 364 1 42 29 80 80 90.40% 7 − 5788785657887907 52 1 42 29 80 80 90.40% 5 − 46235561 46235612 52 1 42 29 80 8090.40% 3 − 93776326 93776377 52 1 42 29 80 80 90.40% 2 − 9452660194526652 52 1 41 32 80 80 91.90% 7 − 57961929 57961977 49 1 41 28 80 8088.70% 12 − 37636906 37636958 53 1 40 33 80 80 91.70% Y − 1061106410611111 48 1 40 29 80 80 88.50% Un_KI270757v1 − 69231 69282 52 1 40 2980 80 88.50% 2 − 94519096 94519147 52 1 40 31 80 80 90.00% 12 − 3734749137347540 50 1 40 29 80 80 88.50% 10 − 39474813 39474864 52 1 39 28 80 8086.80% 12 − 37629404 37629456 53 1 38 29 80 80 86.60% Y − 1022456610224617 52 1 38 33 80 80 89.60% 22_KI270738v1_random − 88363 88410 48 138 33 80 80 89.60% 16 − 34206214 34206261 48 1 38 29 80 80 86.60% 15 −19810704 19810755 52 1 38 29 80 80 86.60% 10 − 39433692 39433743 52 1 3733 79 80 89.40% 12 − 37253870 37253916 47 1 36 37 80 80 91.00% 22 −16010683 16010726 44 1 35 34 80 80 87.30% 5 − 46428661 46428707 47 1 3429 66 80 94.80% 5 − 50180200 50180237 38 1 34 29 66 80 94.80% 2 −94542485 94542522 38 1 33 45 80 80 97.20% 1 − 121859629 121859837 209 132 33 66 80 97.10% 9 − 63089578 63089611 34 1 32 33 66 80 91.00% 7 −61300585 61300617 33 1 32 31 66 80 94.50% 22 − 16077012 16077047 36 1 3233 66 80 97.10% 22 − 15205349 15205382 34 1 32 24 59 80 97.10% 22 −12333177 12333554 378 1 29 50 80 80 96.80% 5 − 49626509 49626539 31 1 2750 80 80 93.60% 3 − 90708676 90708706 31 1 27 45 79 80 88.60% 1 −124993436 124993470 35 1 26 1 30 80 93.40% Un_KI270411v1 − 771 800 30 126 33 64 80 90.70% 22 − 16069657 16069688 32 1 26 51 80 80 93.40% 10 −41806251 41806280 30 1 25 1 29 80 93.20% 3 − 93710289 93710317 29 1 2245 66 80 100.00% 5 − 50205720 50205741 22 10 80 1 80 80 100.00% 3 −93708028 93708107 80 10 80 1 80 80 100.00% 3 − 93710913 93710992 80 1080 1 80 80 100.00% 3 + 91649275 91649354 80 10 80 1 80 80 100.00% 3 +91643494 91643573 80 10 80 1 80 80 100.00% 3 + 91636352 91636431 80 1080 1 80 80 100.00% 3 + 91627682 91627761 80 10 80 1 80 80 100.00% 3 +91624790 91624869 80 10 80 1 80 80 100.00% 3 + 91621897 91621976 80 1080 1 80 80 100.00% 3 + 91619005 91619084 80 10 80 1 80 80 100.00% 3 +91616114 91616193 80 10 80 1 80 80 100.00% 3 + 91610670 91610749 80 1080 1 80 80 100.00% 3 + 91599103 91599182 80 10 80 1 80 80 100.00% 3 +91596212 91596291 80 10 80 1 80 80 100.00% 3 + 91592982 91593061 80 1080 1 80 80 100.00% 3 + 91588048 91588127 80 10 80 1 80 80 100.00% 3 +91573595 91573674 80 10 80 1 80 80 100.00% 3 + 91565255 91565334 80 1080 1 80 80 100.00% 3 + 91559804 91559883 80 10 78 1 80 80 98.80% 3 −91551019 91551098 80 10 76 1 80 80 97.50% 3 − 91542694 91542773 80 10 721 80 80 95.00% 3 − 91544904 91544983 80 10 70 1 80 80 93.80% 3 −91547119 91547198 80 10 64 1 80 80 90.00% 3 − 93714814 93714893 80 10 641 80 80 90.00% 3 − 90575684 90575763 80 10 63 1 80 80 90.00% 3 −90555710 90634361 78652 10 62 1 80 80 88.80% 10 − 41838082 41838161 8010 61 1 80 80 88.80% 3 − 90509626 90532074 22449 10 60 1 80 80 87.50% 3− 90613130 90613209 80 10 60 1 80 80 87.50% 3 − 90600765 90600844 80 1060 1 80 80 87.50% 3 − 90485193 90485272 80 10 59 1 68 80 94.20% 10 −41833865 41840361 6497 10 58 1 80 80 83.60% 3 − 90498916 90498994 79 1052 1 68 80 88.30% 3 − 90638554 90638621 68 10 50 1 68 80 86.80% 3 −90545644 90545711 68 10 48 16 79 80 87.50% 12 − 34712579 34712642 64 1047 1 59 80 91.30% 3 − 90520976 90521034 59 10 47 1 59 80 89.90% 3 −90593213 90593271 59 10 46 1 60 80 88.40% 3 − 90500811 90500870 60 10 4229 80 80 90.40% 21 − 10756848 10756899 52 10 42 29 80 80 90.40% 19 −27333090 27333141 52 10 41 34 80 80 93.70% 22 − 12355283 12355329 47 1040 33 80 80 91.70% 7 − 57969838 57969885 48 10 39 34 80 80 91.50%Un_KI270589v1 − 27295 27341 47 10 39 34 80 80 91.50% Un_KI270579v1 −27288 27334 47 10 39 34 80 80 91.50% 7 − 57960043 57960089 47 10 39 3379 80 91.50% 22 − 16054829 16054875 47 10 39 34 80 80 91.50% 22 −11377572 11377618 47 10 39 34 80 80 91.50% 20 − 29863881 29863927 47 1039 34 80 80 91.50% 11 − 50769658 50769704 47 10 38 35 80 80 91.40%Un_KI270589v1 − 13168 13213 46 10 38 33 80 80 89.60% 2 − 9454912194549168 48 10 37 1 59 80 81.40% 3 − 90508118 90508176 59 10 37 29 73 8091.20% 15 − 19820860 19820904 45 10 36 41 80 80 95.00% Un_KI270579v1 −27628 27667 40 10 36 33 80 80 87.50% 22_KI270738v1_random − 86238 8628548 10 36 40 80 80 95.00% 21 − 10762823 10762863 41 10 36 41 80 80 95.00%21 − 5285438 5285477 40 10 36 33 80 80 87.50% 14 − 18257663 18257710 4810 35 40 80 80 92.70% Un_KI270442v1 − 144873 144913 41 10 35 40 80 8092.70% 9 − 63077148 63077188 41 10 35 40 80 80 92.70% 3 − 9377820393778243 41 10 35 40 80 80 92.70% 22 − 16051073 16051113 41 10 35 40 8080 92.70% 22 − 16052263 16052303 41 10 35 40 80 80 92.70% 22 − 1518486815184908 41 10 35 40 80 80 92.70% 21 − 12985458 12985498 41 10 34 33 7880 87.00% Un_KI270589v1 − 29515 29560 46 10 34 34 73 80 92.50% 22 −12356485 12356524 40 10 34 40 79 80 92.50% 21 − 7225624 7225663 40 10 3342 80 80 92.40% Un_KI270519v1 − 30518 30556 39 10 33 40 80 80 90.30%Un_KI270519v1 − 32383 32423 41 10 33 42 80 80 92.40% Un_KI270442v1 −370163 370201 39 10 33 40 80 80 90.30% 7 − 57975983 57976023 41 10 33 4080 80 90.30% 22 − 12364118 12364158 41 10 33 26 68 80 83.40% 12 −37885575 37885616 42 10 32 40 73 80 97.10% Un_KI270589v1 − 15210 1524334 10 32 42 79 80 92.20% 7 − 57980764 57980801 38 10 32 43 80 80 92.20%21 − 5289218 5289255 38 10 32 45 80 80 94.50% 16 − 36257813 36257848 3610 31 33 73 80 87.90% 22 − 15186415 15186455 41 10 31 27 73 80 83.00% 12− 37929402 37929448 47 10 31 44 80 80 91.90% 11 − 50769318 50769354 3710 30 1 68 80 86.20% 11 − 50778972 50779038 67 10 29 1 59 80 90.40% 3 −93710254 93710311 58 10 29 44 80 80 85.80% 22 − 16063523 16063558 36 1029 42 72 80 96.80% 21 − 5291668 5291698 31 10 29 34 68 80 91.50% 15 −19815145 19815179 35 10 29 26 68 80 89.20% 12 − 37850823 37851037 215 1029 27 73 80 80.90% 12 − 37920020 37920066 47 10 28 42 73 80 93.80% 22 −16009320 16009351 32 10 28 42 73 80 93.80% 21 − 12982905 12982936 32 1028 40 73 80 94.00% 12 − 37268192 37268226 35 10 28 27 68 80 83.40% 12 −37844185 37844226 42 10 27 27 59 80 91.00% 11 − 54933954 54933986 33 1027 29 59 80 93.60% 1 − 124976046 124976076 31 10 26 44 73 80 93.40%22_KI270738v1_random − 88364 88393 30 10 26 27 58 80 90.70% 10 −39298365 39298396 32 10 26 40 73 80 88.30% 1 − 122462724 122462757 34 1025 45 73 80 93.20% 21 − 10758390 10758418 29 10 24 34 59 80 96.20% 20 −28876434 28876459 26 10 23 26 58 80 84.90% 12 − 37876030 37876062 33 1023 43 73 80 87.10% 10 − 41831654 41831684 31 10 21 44 68 80 92.00%22_KI270738v1_random − 86419 86443 25 10 21 44 68 80 92.00% 22 −12353416 12353440 25 10 20 40 59 80 100.00% 3 − 90641959 90641978 20 1020 45 68 80 91.70% 12 − 37853963 37853986 24 11 80 1 80 80 100.00% 3 +91997659 91997738 80 11 80 1 80 80 100.00% 3 + 91982864 91982943 80 1180 1 80 80 100.00% 3 + 91977079 91977158 80 11 80 1 80 80 100.00% 3 +91927923 91928002 80 11 80 1 80 80 100.00% 3 + 91922139 91922218 80 1180 1 80 80 100.00% 3 + 91890743 91890822 80 11 80 1 80 80 100.00% 3 +91854183 91854262 80 11 80 1 80 80 100.00% 3 + 91842613 91842692 80 1180 1 80 80 100.00% 3 + 91836828 91836907 80 11 80 1 80 80 100.00% 3 +91817264 91817343 80 11 80 1 80 80 100.00% 3 + 91802809 91802888 80 1180 1 80 80 100.00% 3 + 91799918 91799997 80 11 80 1 80 80 100.00% 3 +91767768 91767847 80 11 80 1 80 80 100.00% 3 + 91750419 91750498 80 1180 1 80 80 100.00% 3 + 91613639 91613718 80 11 80 1 80 80 100.00% 3 +91602074 91602153 80 11 78 1 80 80 98.80% 3 − 91550939 91551018 80 11 761 80 80 97.50% 3 − 93707948 93708027 80 11 76 1 80 80 97.50% 3 −93710833 93710912 80 11 74 1 80 80 96.30% 3 − 91542614 91542693 80 11 741 80 80 96.30% 3 − 91544824 91544903 80 11 74 1 80 80 96.30% 3 −91547039 91547118 80 11 70 1 80 80 93.80% 10 − 41831907 41831986 80 1168 1 80 80 92.50% 3 − 93714734 93714813 80 11 68 1 80 80 92.50% 3 −93716257 93716336 80 11 68 1 80 80 92.50% 10 − 41835634 41835713 80 1168 1 80 80 92.50% 10 − 41840203 41840282 80 11 64 1 80 80 90.00% 3 −90598981 90599060 80 11 64 1 80 80 90.00% 2 − 94527370 94527449 80 11 632 80 80 89.90% 22 − 15161071 15161149 79 11 63 1 80 80 90.00% 20 −29701711 29713360 11650 11 62 2 80 80 89.80% 3 − 90621526 90621946 42111 62 1 80 80 88.80% 3 − 90496461 90496540 80 11 62 1 80 80 88.80% 22 −16036304 16036383 80 11 62 1 80 80 88.80% 22 − 15163800 15163879 80 1162 1 80 80 88.80% 2 − 94551261 94551340 80 11 62 1 80 80 89.80% 11 −55254796 55255047 252 11 62 1 80 80 88.80% 11 − 48915432 48915511 80 1161 1 80 80 89.70% 2 − 94517816 94518065 250 11 61 1 80 80 88.80% 11 −55256150 55257431 1282 11 60 1 80 80 87.50% 3 − 90449684 90449763 80 1160 1 80 80 87.50% 22 − 15167211 15167290 80 11 60 1 80 80 87.50% 20 −29704267 29704346 80 11 60 1 80 80 87.50% 2 − 94525985 94526064 80 11 601 80 80 87.50% 14 − 18244779 18244858 80 11 59 1 80 80 87.50% 11 −48921371 48933809 12439 11 59 1 80 80 87.50% 1 − 124995731 1249975171787 11 59 1 80 80 88.40% 1 − 124976116 124976195 80 11 58 1 80 8086.30% 5 − 47091166 47091245 80 11 58 1 80 80 86.30% 3 − 9049815690498235 80 11 58 1 80 80 86.30% 3 − 90509546 90509625 80 11 58 1 80 8086.30% 3 − 90456136 90456215 80 11 58 1 80 80 86.30% 22 − 1603992616040005 80 11 58 1 80 80 86.30% 2 − 94523090 94523169 80 11 57 1 80 8088.00% 5_GL000208v1_random − 47241 47491 251 11 57 2 80 80 86.10% 22 −16039071 16039149 79 11 57 1 80 80 86.10% 21 − 12965841 12966262 422 1156 1 80 80 85.00% 3 − 91538541 91538620 80 11 56 1 80 80 85.00% 3 −90405456 90405535 80 11 56 1 80 80 85.00% 22_KI270739v1_random − 4540445483 80 11 56 1 80 80 85.00% 22 − 16001756 16001835 80 11 56 1 80 8085.00% 17 − 26880421 26880500 80 11 56 1 80 80 85.00% 11 − 4891369648913775 80 11 55 2 80 80 84.90% 20 − 30979467 30979545 79 11 55 18 8080 93.70% 10 − 41833773 41833835 63 11 54 1 80 80 83.80% 5 − 4708641647086495 80 11 54 1 80 80 83.80% 22 − 15175916 15175995 80 11 54 2 80 8088.60% 14 − 18228743 18229162 420 11 54 1 80 80 83.80% 11 − 5524763155247710 80 11 54 1 80 80 91.00% 11 − 50767708 50767954 247 11 53 1 8080 88.60% Un_KI270442v1 − 121500 122916 1417 11 53 1 80 80 89.60% 5 −46332980 46333399 420 11 53 2 80 80 88.50% 3 − 91523506 91523924 419 1153 1 80 80 83.80% 3 − 90403921 90499425 95505 11 53 2 80 80 83.60% 14 −18239661 18239739 79 11 53 1 80 80 83.80% 1 − 124958321 124959254 934 1152 1 80 80 82.50% 11 − 55258036 55258115 80 11 51 18 80 80 90.50% 3 −91527761 91527823 63 11 51 20 80 80 91.90% 3 − 90577982 90578042 61 1151 18 80 80 90.50% 22_KI270739v1_random − 59626 59688 63 11 51 18 80 8090.50% 22 − 16032546 16032608 63 11 51 2 80 80 83.10% 22 − 1518923115189308 78 11 51 18 80 80 90.50% 22 − 15169776 15169838 63 11 50 19 8080 90.40% 5 − 46418401 46418462 62 11 50 21 80 80 91.70%22_KI270739v1_random − 56551 56610 60 11 50 1 80 80 81.30% 20 − 3098474130984820 80 11 50 1 60 80 91.70% 12 − 34710651 34710710 60 11 49 18 8080 88.90% 3 − 93765881 93765943 63 11 49 18 80 80 88.90% 22 − 1301011113010173 63 11 49 18 80 80 88.90% 2 − 94533001 94533063 63 11 49 18 8080 88.90% 14 − 16129316 16129378 63 11 49 18 80 80 88.90% 1 − 124979346124979408 63 11 47 18 80 80 87.40% 11 − 55249851 55249913 63 11 46 21 8080 88.40% 22 − 16041446 16041505 60 11 46 1 60 80 88.40% 22 − 1515904815159107 60 11 45 20 80 80 86.90% 20 − 29698978 29699038 61 11 43 26 8080 89.10% 9 − 60644999 60645053 55 11 41 18 80 80 82.60% Un_KI270757v1 −59726 59788 63 11 41 28 80 80 88.70% 1 − 124986851 124986903 53 11 40 1880 80 93.50% 11 − 55233465 55233868 404 11 39 1 47 80 91.50%Un_KI270422v1 − 666 712 47 11 39 33 80 80 91.70% 2 − 94516796 94517014219 11 36 2 45 80 91.00% 20 − 30980850 30980893 44 11 35 18 80 80 89.20%20 − 30996656 30996716 61 11 34 1 47 80 87.50% 11 − 48936499 48936544 4611 29 2 38 80 89.20% 5 − 46361577 46361613 37 11 24 49 80 80 87.50% 5 −46361706 46361737 32 11 23 18 42 80 96.00% 16 − 34612003 34612027 25 1122 1 34 80 82.40% 22 − 15957342 15957375 34 11 20 57 80 80 91.70% 20 −29713110 29713133 24 11 20 28 47 80 100.00% 2 − 94550097 94550116 20 1280 1 80 80 100.00% 3 − 93713482 93713561 80 12 80 1 80 80 100.00% 3 −91544583 91544662 80 12 80 1 80 80 100.00% 3 − 91550699 91550778 80 1280 1 80 80 100.00% 3 + 91681077 91681156 80 12 80 1 80 80 100.00% 3 +91675297 91675376 80 12 80 1 80 80 100.00% 3 + 91669512 91669591 80 1280 1 80 80 100.00% 3 + 91652493 91652572 80 12 80 1 80 80 100.00% 3 +91643814 91643893 80 12 80 1 80 80 100.00% 3 + 91636672 91636751 80 1280 1 80 80 100.00% 3 + 91628002 91628081 80 12 80 1 80 80 100.00% 3 +91613879 91613958 80 12 80 1 80 80 100.00% 3 + 91605208 91605287 80 1280 1 80 80 100.00% 3 + 91599423 91599502 80 12 80 1 80 80 100.00% 3 +91596532 91596611 80 12 80 1 80 80 100.00% 3 + 91593302 91593381 80 1280 1 80 80 100.00% 3 + 91590407 91590486 80 12 80 1 80 80 100.00% 3 +91588368 91588447 80 12 80 1 80 80 100.00% 3 + 91582583 91582662 80 1280 1 80 80 100.00% 3 + 91576802 91576881 80 12 78 1 80 80 98.80% 3 −93710593 93710672 80 12 78 1 80 80 98.80% 3 − 93707708 93707787 80 12 781 80 80 98.80% 3 − 91546798 91546877 80 12 72 1 80 80 95.00% 3 −93715345 93715424 80 12 68 1 80 80 92.50% 12 − 37265167 37265246 80 1265 1 80 80 91.30% 12 − 34718537 34834136 115600 12 64 2 80 80 91.20% 3 −93707033 93707278 246 12 64 2 80 80 91.20% 3 − 93712467 93712713 247 1264 2 80 80 91.20% 12 − 34716850 34717093 244 12 63 1 80 80 90.00% 12 −34717858 34718617 760 12 62 1 80 80 88.80% 5 − 49610180 49610259 80 1262 1 80 80 88.80% 5 − 49631535 49631614 80 12 62 2 80 80 89.90% 3 −91541705 91541944 240 12 62 1 80 80 88.80% 10 − 41840813 41840892 80 1261 1 74 80 91.90% 12 − 37251118 37254019 2902 12 61 8 80 80 91.80% 12 −37263475 37263547 73 12 61 6 80 80 90.70% 12 − 34717522 34717596 75 1260 1 80 80 87.50% Un_KI270317v1 − 1492 1571 80 12 60 1 80 80 87.50%Un_KI270317v1 − 37214 37293 80 12 60 1 80 80 87.50% 5 − 4962346549623544 80 12 60 1 80 80 87.50% 5 − 49625504 49625583 80 12 60 1 76 8089.50% 5 − 49611202 49611277 76 12 60 1 80 80 87.50% 4 − 6739979667399875 80 12 60 2 80 80 88.70% 3 − 91552909 91553154 246 12 60 1 76 8089.50% 16 − 38280320 38280395 76 12 60 1 71 80 93.00% 12 − 3725462937257395 2767 12 60 8 75 80 91.10% 12 − 37262797 37262863 67 12 60 1 7480 90.60% 12 − 34832649 34832722 74 12 59 1 80 80 94.10% 12 − 3725867137258806 136 12 59 1 72 80 91.70% 12 − 34832991 34833171 181 12 58 1 7480 89.20% 12 − 34830733 34830806 74 12 57 8 74 80 92.60% 12 − 3726653137266597 67 12 56 1 80 80 85.00% Un_KI270312v1 − 38 117 80 12 56 1 74 8087.90% 12 − 34829375 34829448 74 12 54 1 76 80 85.60% 16 − 3827930638279381 76 12 53 8 72 80 90.80% 4 − 67398785 67398849 65 12 53 2 80 8083.60% 3 − 90360495 90360573 79 12 52 5 76 80 86.20% 14 − 1824812518248196 72 12 50 5 80 80 82.90% 5 − 46357746 46357821 76 12 50 5 80 8082.90% 12 − 37671976 37672051 76 12 50 5 80 80 82.90% 11 − 5078095350781028 76 12 49 5 80 80 82.90% 3 − 90287633 90578158 290526 12 49 6 7280 86.60% 12 − 37260434 37260500 67 12 49 2 80 80 81.10% 12 − 3769402637694104 79 12 49 2 76 80 82.70% 11 − 50775330 50775404 75 12 49 2 80 8081.10% 11 − 50729863 50729941 79 12 49 2 80 80 81.10% 10 − 3948942139489499 79 12 48 2 69 80 85.30% 3 − 90643159 90643226 68 12 48 11 76 8086.40% 16 − 38268796 38268861 66 12 48 5 80 80 91.40% 14 − 1823157018231816 247 12 48 1 80 80 88.80% 14 − 16129246 16129496 251 12 47 2 8080 79.80% Un_KI270442v1 − 207790 207868 79 12 46 5 80 80 80.30% 14 −16127416 16127491 76 12 46 5 76 80 82.00% 12 − 37675727 37675798 72 1246 2 80 80 91.10% 10 − 39439308 39473539 34232 12 45 5 73 80 88.70% 3 −90672344 90672411 68 12 45 2 80 80 78.50% 12 − 37673165 37673243 79 1245 2 80 80 78.50% 11 − 55233907 55233985 79 12 44 5 72 80 82.40%Un_KI270411v1 − 1761 1828 68 12 43 2 80 80 95.80% 10 − 39473461 3948489211432 12 42 2 57 80 87.50% 5 − 46431426 46431481 56 12 41 5 80 80 91.90%3 − 93710254 93710497 244 12 41 5 73 80 79.80% 3 − 90519625 90519693 6912 41 2 75 80 90.20% 11 − 48760140 48760214 75 12 37 5 57 80 85.00% 3 −90653859 90653911 53 12 37 2 80 80 95.20% 14 − 18268603 18268852 250 1237 2 60 80 81.40% 12 − 37702055 37702113 59 12 36 2 57 80 97.40% 21 −12996456 12996682 227 12 36 1 80 80 97.40% 20 − 28876264 28876343 80 1235 2 80 80 97.30% 21 − 7222919 7222997 79 12 35 2 80 80 97.30% 11 −50779589 50779667 79 12 35 2 59 80 95.00% 11 − 50726716 50726773 58 1235 5 57 80 83.10% 11 − 50799784 50799836 53 12 32 2 57 80 78.60% 10 −41839819 41839874 56 12 31 44 80 80 91.90% 5 − 50198221 50198257 37 1229 44 80 80 89.20% 16 − 34612237 34612273 37 12 29 44 80 80 89.20% 11 −48915191 48915227 37 12 28 44 80 80 96.70% 21 − 5297271 5297478 208 1227 1 27 80 100.00% 3 − 93705728 93705754 27 12 27 44 74 80 93.60% 11 −55251142 55251172 31 12 26 2 27 80 100.00% 3 − 90636069 90636094 26 1224 2 25 80 100.00% 15 − 19813844 19813867 24 12 23 5 27 80 100.00% 3 −90674431 90674453 23 12 23 5 27 80 100.00% 3 − 90683359 90683381 23 1222 57 80 80 95.90% Un_KI270317v1 − 473 496 24 12 22 2 25 80 95.90% 14 −16082967 16082990 24 12 22 2 25 80 95.90% 11 − 50777601 50777624 24 1221 5 25 80 100.00% 7 − 62572906 62572926 21 13 80 1 80 80 100.00% 3 +91782552 91782631 80 13 80 1 80 80 100.00% 3 + 91776766 91776845 80 1380 1 80 80 100.00% 3 + 91744957 91745036 80 13 80 1 80 80 100.00% 3 +91739515 91739594 80 13 80 1 80 80 100.00% 3 + 91663810 91663889 80 1380 1 80 80 100.00% 3 + 91655466 91655545 80 13 80 1 80 80 100.00% 3 +91649675 91649754 80 13 80 1 80 80 100.00% 3 + 91646786 91646865 80 1380 1 80 80 100.00% 3 + 91641001 91641080 80 13 80 1 80 80 100.00% 3 +91633861 91633940 80 13 80 1 80 80 100.00% 3 + 91628082 91628161 80 1380 1 80 80 100.00% 3 + 91596612 91596691 80 13 80 1 80 80 100.00% 3 +91585556 91585635 80 13 80 1 80 80 100.00% 3 + 91579771 91579850 80 1380 1 80 80 100.00% 3 + 91576882 91576961 80 13 80 1 80 80 100.00% 3 +91565655 91565734 80 13 78 1 80 80 98.80% 3 − 91550619 91550698 80 13 772 80 80 98.80% 3 − 91544503 91544581 79 13 74 1 80 80 96.30% 3 −93713402 93713481 80 13 74 1 80 80 96.30% 3 − 93710513 93710592 80 13 721 80 80 95.00% 3 − 93707628 93707707 80 13 72 1 80 80 92.50% 3 −91546719 91546797 79 13 65 1 80 80 91.20% 3 − 93711368 93715674 4307 1364 1 80 80 90.00% 12 − 37265427 37265506 80 13 64 1 80 80 90.00% 12 −37263735 37263814 80 13 63 1 80 80 90.00% 12 − 37239490 37239909 420 1362 1 80 80 88.80% 5 − 49643720 49643799 80 13 62 1 80 80 88.80% 5 −49626444 49626523 80 13 62 1 80 80 88.80% 3 − 91552490 91552569 80 13 621 80 80 88.80% 16 − 38265990 38266069 80 13 62 1 80 80 88.80% 16 −38276169 38276248 80 13 62 1 80 80 88.80% 12 − 37261018 37261097 80 1362 1 80 80 88.80% 12 − 34820817 34820896 80 13 62 1 80 80 88.80% 12 −34820478 34820557 80 13 62 1 80 80 88.80% 10 − 41839549 41839628 80 1360 1 80 80 87.50% 5 − 49645410 49645489 80 13 60 1 80 80 87.50% 5 −49645075 49645154 80 13 60 1 80 80 87.50% 5 − 49642370 49642449 80 13 601 80 80 87.50% 5 − 49624745 49624824 80 13 60 1 80 80 87.50% 16 −38276509 38276588 80 13 60 1 80 80 87.50% 12 − 37266785 37266864 80 1360 1 80 80 87.50% 10 − 41833965 41834044 80 13 58 1 80 80 86.30% 5 −49623725 49623804 80 13 58 1 80 80 86.30% 12 − 37252445 37252524 80 1357 12 80 80 91.40% 3 − 90520385 90520453 69 13 57 12 80 80 91.40% 12 −34716770 34716838 69 13 57 12 80 80 91.40% 10 − 41835826 41835894 69 1356 1 80 80 85.00% 5 − 49624405 49624484 80 13 56 1 80 80 85.00% 12 −37240170 37240249 80 13 55 12 80 80 89.90% Un_KI270317v1 − 36455 3652369 13 55 12 80 80 89.90% 5 − 49622710 49622778 69 13 55 12 80 80 89.90%3 − 93710174 93710242 69 13 55 12 80 80 89.90% 3 − 93713063 93713131 6913 55 12 80 80 89.90% 3 − 91550280 91550348 69 13 55 12 80 80 89.90% 3 −91549603 91549671 69 13 55 12 80 80 89.90% 3 − 91544163 91544231 69 1355 12 80 80 89.90% 3 − 90533635 90533703 69 13 53 12 80 80 88.50% 3 −91282819 91282887 69 13 53 12 80 80 88.50% 3 − 91281485 91281553 69 1353 12 80 80 88.50% 3 − 90684585 90684653 69 13 53 12 80 80 88.50% 3 −90678209 90678277 69 13 53 12 80 80 88.50% 3 − 90619678 90619746 69 1353 12 80 80 88.50% 3 − 90576134 90576202 69 13 53 12 80 80 88.50% 12 −37261698 37261766 69 13 53 12 80 80 88.50% 12 − 34716089 34716157 69 1352 12 79 80 88.30% 5 − 49642711 49642778 68 13 52 13 80 80 88.30% 3 −90605289 90605356 68 13 52 21 80 80 93.40% 12 − 34713030 34713089 60 1351 12 80 80 87.00% Un_KI270329v1 − 860 928 69 13 51 12 80 80 87.00% 5 −49643045 49643113 69 13 51 20 80 80 91.90% 3 − 93705595 93705655 61 1351 20 80 80 91.90% 3 − 93708483 93708543 61 13 50 19 80 80 90.40%Un_KI270411v1 − 318 379 62 13 50 21 80 80 91.70% 12 − 37270326 3727038560 13 50 21 80 80 91.70% 12 − 34714054 34714113 60 13 50 19 80 80 90.40%10 − 41840733 41840794 62 13 49 18 80 80 88.90% 4 − 67398697 67398759 6313 49 18 80 80 88.90% 12 − 34719135 34719197 63 13 49 20 80 80 90.20% 10− 41838194 41838254 61 13 48 19 80 80 96.20% Un_KI270411v1 − 1335 1734400 13 48 22 80 80 91.40% 7 − 60884192 60884579 388 13 48 1 60 80 90.00%5 − 49610120 49610179 60 13 48 21 80 80 90.00% 12 − 34710310 34710369 6013 46 23 80 80 89.70% 12 − 34711334 34711391 58 13 45 22 80 80 88.20%Un_KI270366v1 − 3484 3542 59 13 45 22 80 80 88.20% 5_GL000208v1_random −46239 46297 59 13 44 21 80 80 86.70% 12 − 34715075 34715134 60 13 44 2580 80 89.30% 10 − 41834305 41834360 56 13 43 22 80 80 86.50% 2 −94509270 94509328 59 13 34 45 80 80 97.30% 12 − 37251709 37251744 36 1334 45 80 80 97.30% 12 − 34834994 34835029 36 13 33 44 80 80 94.60% 3 −93715265 93715301 37 13 33 44 80 80 94.60% 12 − 34709971 34710007 37 1333 21 80 80 97.20% 10 − 41832264 41832323 60 13 29 48 80 80 94.00% 5 −49617630 49617662 33 13 27 52 80 80 96.60% Un_KI270312v1 − 299 327 29 1327 52 80 80 96.60% 5 − 49642030 49642058 29 13 27 49 80 80 93.60% 2 −94522436 94522468 33 13 24 53 80 80 92.90% 5_GL000208v1_random − 4794447971 28 13 21 21 41 80 100.00% 12 − 34709672 34709692 21 14 78 1 80 8096.30% 3 − 91550540 91550618 79 14 78 1 80 80 96.30% 3 + 9162816291628240 79 14 78 1 80 80 96.30% 3 + 91625270 91625348 79 14 78 1 80 8096.30% 3 + 91622377 91622455 79 14 78 1 80 80 96.30% 3 + 9161948691619564 79 14 78 1 80 80 96.30% 3 + 91614040 91614118 79 14 78 1 80 8096.30% 3 + 91611150 91611228 79 14 78 1 80 80 96.30% 3 + 9159669291596770 79 14 78 1 80 80 96.30% 3 + 91593462 91593540 79 14 78 1 80 8096.30% 3 + 91585636 91585714 79 14 78 1 80 80 96.30% 3 + 9157985191579929 79 14 78 1 80 80 96.30% 3 + 91576962 91577040 79 14 78 1 80 8096.30% 3 + 91574075 91574153 79 14 78 1 80 80 96.30% 3 + 9157118191571259 79 14 78 1 80 80 96.30% 3 + 91568630 91568708 79 14 78 1 80 8096.30% 3 + 91565735 91565813 79 14 78 1 80 80 96.30% 3 + 9156284191562919 79 14 76 1 80 80 95.00% 3 − 91544424 91544502 79 14 74 1 80 8093.70% 3 − 93713323 93713401 79 14 74 1 80 80 93.70% 3 − 9371043493710512 79 14 72 1 80 80 92.50% 3 − 93707549 93707627 79 14 63 12 80 8095.70% 3 − 93707890 93707958 69 14 63 12 80 80 95.70% 3 − 9371077593710843 69 14 63 12 80 80 95.70% 3 − 91550881 91550949 69 14 63 12 8080 95.70% 3 − 91542556 91542624 69 14 63 12 80 80 95.70% 10 − 4183980841839876 69 14 62 12 80 80 97.00% 10 − 41831509 41831917 409 14 61 12 8080 94.30% 3 − 93706874 93706942 69 14 61 12 80 80 94.30% 3 − 9370975793709825 69 14 61 12 80 80 94.30% 3 − 93722329 93722397 69 14 61 12 8080 94.30% 3 − 93723519 93723587 69 14 61 12 80 80 94.30% 3 − 9155275091552818 69 14 61 12 80 80 94.30% 3 − 91549863 91549931 69 14 61 12 8080 94.30% 3 − 91544766 91544834 69 14 61 12 80 80 94.30% 3 − 9050609590506163 69 14 60 12 80 80 95.60% X − 62483966 62494319 10354 14 60 1280 80 95.60% X − 62494251 62500490 6240 14 60 12 80 80 95.60% X −62477796 62484034 6239 14 60 12 80 80 95.60% X − 62469571 62477864 829414 60 12 80 80 95.60% X − 62465456 62469639 4184 14 60 12 80 80 94.30%10 − 41834900 41835644 745 14 59 12 80 80 92.80% 3 − 91546981 9154704969 14 59 12 80 80 92.80% 3 − 91518720 91518788 69 14 59 12 80 80 92.80%3 − 90597560 90597628 69 14 57 12 80 80 91.40% 3 − 93717220 93717288 6914 57 12 80 80 91.40% 3 − 93734545 93734613 69 14 57 12 80 80 91.40% 3 −91526340 91526408 69 14 57 12 80 80 91.40% 3 − 91262029 91262097 69 1457 12 80 80 91.40% 3 − 90474146 90474214 69 14 57 12 80 80 91.40% 3 −90443156 90443224 69 14 57 12 80 80 91.40% 22 − 15176371 15176439 69 1457 12 80 80 92.80% 11 − 48931965 48932204 240 14 57 12 80 80 91.40% 11 −48926781 48926849 69 14 57 12 80 80 91.40% 1 − 121613914 121613982 69 1456 17 80 80 93.80% 3 − 90543072 90543135 64 14 56 1 80 80 84.90% 12 −34715676 34715750 75 14 56 12 80 80 92.50% 11 − 48922681 48922750 70 1455 13 80 80 91.20% 9 − 60613301 60613369 69 14 55 12 80 80 89.90%5_GL000208v1_random − 29509 29577 69 14 55 12 80 80 89.90% 5 − 4641953646419604 69 14 55 12 80 80 92.40% 22 − 15175858 15176097 240 14 55 12 8080 89.90% 2 − 94533456 94533524 69 14 55 12 80 80 89.90% 17 − 2688291426882982 69 14 55 12 80 80 89.90% 15 − 19794091 19794159 69 14 54 12 8080 93.70% 12 − 37254121 37254189 69 14 53 21 80 80 95.00% 22 − 1599811515998345 231 14 53 12 80 80 88.50% 20 − 30157810 30157878 69 14 53 12 8080 88.50% 2 − 94506291 94506359 69 14 53 17 80 80 95.00% 12 − 3723941537239645 231 14 53 12 80 80 88.50% 11 − 48933331 48933399 69 14 52 17 8080 90.70% 9 − 60598511 60598574 64 14 52 17 80 80 90.70% 9 − 6060650160606564 64 14 52 17 80 80 90.70% 9 − 60607691 60607754 64 14 52 17 8080 90.70% 9 − 60622651 60622714 64 14 52 17 80 80 90.70% 9 − 6063796660638029 64 14 52 21 80 80 93.40% 14 − 18238235 18238294 60 14 52 21 8080 93.40% 14 − 18240971 18241030 60 14 51 12 80 80 87.00% 9 − 6058966160589729 69 14 51 12 80 80 87.00% 5 − 47084821 47084889 69 14 51 12 8080 87.00% 12 − 34831586 34831654 69 14 50 17 80 80 89.10% 9 − 6057877160578834 64 14 50 17 80 80 89.10% 9 − 60604121 60604184 64 14 50 17 8080 85.80% 5 − 47090596 47090658 63 14 50 12 80 80 93.20% 21 − 1296846612968705 240 14 50 17 80 80 89.10% 17 − 25919191 25919254 64 14 49 12 8080 85.60% Un_KI270544v1 − 436 504 69 14 47 12 80 80 89.90%5_GL000208v1_random − 15126 15194 69 14 47 12 80 80 84.10%5_GL000208v1_random − 18760 18828 69 14 47 12 80 80 84.10% 5 − 4709540147095469 69 14 47 12 80 80 84.10% 2 − 94505266 94505334 69 14 46 12 8080 92.50% 2 − 94558879 94559288 410 14 45 31 80 80 96.00% X − 5854730658547541 236 14 44 32 80 80 97.90% X − 62500421 62500656 236 14 43 12 5680 97.80% 12 − 37687878 37687922 45 14 43 12 58 80 95.80% 11 − 5525817155258217 47 14 42 37 80 80 97.80% 12 − 34829550 34829593 44 14 41 17 5880 100.00% 21 − 12970366 12970578 213 14 40 37 80 80 95.50% 12 −37238395 37238438 44 14 40 37 80 80 95.50% 12 − 37239751 37239794 44 1440 37 80 80 95.50% 12 − 37240771 37240814 44 14 38 35 80 80 91.40% 5 −49622631 49622676 46 14 36 35 80 80 89.20% Un_KI270366v1 − 6813 6858 4614 36 17 56 80 95.00% 22_KI270739v1_random − 31269 31308 40 14 31 17 4880 100.00% 22 − 15163093 15163466 374 15 80 1 80 80 100.00% 3 − 9371224293712321 80 15 80 1 80 80 100.00% 3 − 93706469 93706548 80 15 80 1 80 80100.00% 3 + 91685209 91685288 80 15 80 1 80 80 100.00% 3 + 9167075591670834 80 15 80 1 80 80 100.00% 3 + 91664971 91665050 80 15 80 1 80 80100.00% 3 + 91659524 91659603 80 15 80 1 80 80 100.00% 3 + 9165662891656707 80 15 80 1 80 80 100.00% 3 + 91653734 91653813 80 15 80 1 80 80100.00% 3 + 91639610 91639689 80 15 80 1 80 80 100.00% 3 + 9163213191632210 80 15 80 1 80 80 100.00% 3 + 91609339 91609418 80 15 80 1 80 80100.00% 3 + 91606449 91606528 80 15 80 1 80 80 100.00% 3 + 9159165191591730 80 15 80 1 80 80 100.00% 3 + 91583825 91583904 80 15 80 1 80 80100.00% 3 + 91578043 91578122 80 15 80 1 80 80 100.00% 3 + 9157226491572343 80 15 80 1 80 80 100.00% 3 + 91563923 91564002 80 15 80 1 80 80100.00% 3 + 91558474 91558553 80 15 78 1 80 80 98.80% 3 − 9154945891549537 80 15 78 1 80 80 96.30% 3 + 93142761 93142839 79 15 78 1 80 8096.30% 3 + 93208255 93208333 79 15 76 1 80 80 97.50% 3 − 9154401891544097 80 15 75 1 80 80 97.50% 3 − 91552346 91552763 418 15 74 5 80 8096.00% 3 − 93709357 93709431 75 15 73 1 80 80 96.30% 3 − 9154691591548518 1604 15 72 1 80 80 96.30% 3 − 91544700 91546994 2295 15 62 1 8080 87.20% 3 − 93707825 93707903 79 15 62 1 80 80 87.20% 3 − 9371071093710788 79 15 62 1 80 80 87.20% 3 − 91550816 91550894 79 15 62 1 80 8088.80% 3 − 91551327 91551406 80 15 57 1 80 80 89.20% 3 − 9154181991544779 2961 15 51 1 74 80 89.60% 4 − 67399917 67400330 414 15 50 1 8080 83.40% 12 − 37236973 37237050 78 15 50 1 80 80 82.70% 12 − 3483377834833855 78 15 49 1 80 80 82.40% 12 − 37254397 37254474 78 15 49 1 80 8083.10% 12 − 37262573 37262650 78 15 48 1 80 80 81.20% 5 − 4964391349643990 78 15 48 1 80 80 81.20% 5 − 49622567 49622644 78 15 48 1 80 8081.20% 5 − 49618507 49618584 78 15 48 1 80 80 81.20% 5 − 4961544849615525 78 15 48 2 80 80 82.40% 3 − 91545556 91545633 78 15 48 1 80 8081.20% 16 − 38267888 38267965 78 15 48 1 80 80 86.80% 12 − 3726359237265025 1434 15 48 1 80 80 81.90% 12 − 37240367 37240444 78 15 48 1 8080 81.90% 12 − 37239011 37239088 78 15 48 1 80 80 81.90% 12 − 3723561337235690 78 15 47 1 80 80 80.90% Un_KI270317v1 − 36652 36729 78 15 47 180 80 81.60% 12 − 37264608 37264685 78 15 46 1 80 80 79.80%Un_KI270304v1 − 1538 1615 78 15 46 1 80 80 80.40% 12 − 37238331 3723840878 15 45 1 80 80 79.50% 5 − 49618847 49618924 78 15 45 1 80 80 80.00% 5− 49617148 49617225 78 15 43 1 80 80 78.00% 5 − 49645267 49645344 78 1543 1 80 80 78.00% 5 − 49643237 49643314 78 15 43 12 80 80 82.80% 5 −49616468 49616534 67 15 43 12 80 80 82.80% 5 − 49615788 49615854 67 1543 12 80 80 82.80% 12 − 37260203 37260269 67 15 41 12 80 80 81.10% 5 −49623582 49623648 67 15 41 1 55 80 87.30% 12 − 34831545 34831599 55 1540 12 80 80 81.50% 5 − 49624262 49624328 67 15 37 1 60 80 93.10% 3 −91546934 91550214 3281 15 36 12 55 80 91.00% 3 − 93715815 93715858 44 1535 15 80 80 78.50% Un_KI270304v1 − 1198 1261 64 15 35 1 60 80 90.70% 3 −91544719 91546649 1931 15 24 1 26 80 96.20% 3 − 90650781 90650806 26 1524 1 26 80 96.20% 3 − 90504411 90504436 26 15 22 1 26 80 92.40%Un_KI270317v1 − 36364 36389 26 15 22 1 26 80 92.40% Un_KI270310v1 − 850875 26 15 22 1 26 80 92.40% Un_KI270304v1 − 910 935 26 15 22 1 26 8092.40% 3 − 93707200 93707225 26 15 22 1 26 80 92.40% 3 − 9059431990594344 26 15 22 1 26 80 92.40% 12 − 37267374 37267399 26 15 22 1 26 8092.40% 12 − 37259915 37259940 26 15 20 27 46 80 100.00% 8 − 7792762577927644 20 15 20 1 26 80 88.50% 3 − 93713990 93714015 26 15 20 1 26 8088.50% 3 − 90492644 90492669 26 15 20 27 46 80 100.00% 2 − 123296087123296106 20 15 20 3 26 80 91.70% 12 − 34719384 34719407 24 16 78 1 8080 98.80% 3 + 91664456 91664535 80 16 78 1 80 80 98.80% 3 + 9165321991653298 80 16 78 1 80 80 98.80% 3 + 91631616 91631695 80 16 78 1 80 8098.80% 3 + 91628728 91628807 80 16 78 1 80 80 98.80% 3 + 9162583691625915 80 16 78 1 80 80 98.80% 3 + 91622943 91623022 80 16 78 1 80 8098.80% 3 + 91611716 91611795 80 16 78 1 80 80 98.80% 3 + 9160882491608903 80 16 78 1 80 80 98.80% 3 + 91605934 91606013 80 16 78 1 80 8098.80% 3 + 91600149 91600228 80 16 78 1 80 80 98.80% 3 + 9159725891597337 80 16 78 1 80 80 98.80% 3 + 91594028 91594107 80 16 78 1 80 8098.80% 3 + 91591136 91591215 80 16 78 1 80 80 98.80% 3 + 9158909391589172 80 16 78 1 80 80 98.80% 3 + 91574641 91574720 80 16 78 1 80 8098.80% 3 + 91569196 91569275 80 16 76 1 80 80 97.50% 3 − 9154997391550052 80 16 72 1 80 80 95.00% 3 − 93713772 93713851 80 16 72 1 80 8095.00% 3 − 91552860 91552939 80 16 65 12 80 80 97.20% 3 − 9370698493707052 69 16 65 12 80 80 97.20% 3 − 93709867 93709935 69 16 65 12 8080 97.20% 3 − 93712757 93712825 69 16 62 9 80 80 90.20% 10 − 4184144341841513 71 16 62 9 80 80 93.10% 10 − 41839918 41839989 72 16 60 9 80 8091.70% 5 − 49624776 49624847 72 16 60 9 80 80 91.70% 3 − 9154317891543249 72 16 59 10 80 80 91.60% 3 − 90606335 90606405 71 16 59 12 8080 92.80% 12 − 37258622 37258690 69 16 58 9 80 80 90.30% 3 − 9370851493708585 72 16 58 9 80 80 90.30% 3 − 93711399 93711470 72 16 58 9 80 8090.30% 3 − 91545390 91545461 72 16 58 13 80 80 92.70% 12 − 3724631337246380 68 16 58 13 80 80 92.70% 12 − 37239861 37239928 68 16 58 13 8080 92.70% 12 − 37238505 37238572 68 16 58 13 80 80 92.70% 12 − 3723782737237894 68 16 58 13 80 80 92.70% 12 − 37236467 37236534 68 16 58 13 8080 92.70% 12 − 34831017 34831084 68 16 58 9 80 80 90.30% 12 − 3471578234715853 72 16 58 9 80 80 90.30% 10 − 41832130 41832201 72 16 57 10 8080 90.20% 5 − 46418963 46419033 71 16 57 10 80 80 90.20% 3 − 9070915290709222 71 16 57 10 80 80 90.20% 3 − 90683428 90683498 71 16 57 12 8080 91.40% 3 − 90509598 90509666 69 16 57 10 80 80 90.20% 3 − 9050755390507623 71 16 57 10 80 80 90.20% 3 − 90501616 90501686 71 16 57 10 8080 90.20% 3 − 90494473 90494543 71 16 56 9 80 80 88.90% 3 − 9155150591551576 72 16 56 9 80 80 88.90% 3 − 91520000 91520071 72 16 56 11 80 8090.00% 12 − 37257943 37258012 70 16 56 13 80 80 91.20% 12 − 3482966034829727 68 16 56 13 80 80 91.20% 12 − 34822207 34822274 68 16 56 13 8080 91.20% 12 − 34821867 34821934 68 16 56 13 80 80 91.20% 10 − 4184076441840831 68 16 55 10 80 80 88.80% Un_KI270366v1 − 7425 7495 71 16 55 1080 80 88.80% 5_GL000208v1_random − 56663 56733 71 16 55 10 80 80 88.80%3 − 90564638 90564708 71 16 55 10 80 80 88.80% 3 − 90559218 90559288 7116 55 10 80 80 88.80% 3 − 90498208 90498278 71 16 55 17 80 80 93.80% 12− 37263766 37263830 65 16 55 13 77 80 92.40% 12 − 37260040 37260104 6516 55 18 80 80 93.70% 12 − 37252136 37252198 63 16 55 18 80 80 93.70% 10− 41839580 41839642 63 16 54 18 77 80 95.00% 12 − 37261052 37261111 6016 54 18 80 80 93.70% 12 − 34716060 34716182 123 16 53 17 80 80 92.20%12 − 37235447 37235511 65 16 53 17 80 80 92.20% 12 − 37236807 3723687165 16 53 18 80 80 92.10% 12 − 37260713 37260775 63 16 53 18 80 80 92.10%12 − 37259018 37259080 63 16 53 17 78 80 93.60% 12 − 34831358 3483142063 16 52 13 80 80 90.70% 12 − 34710341 34710750 410 16 51 18 80 8090.50% 2 − 94532711 94532773 63 16 51 17 80 80 90.70% 12 − 3723884537238909 65 16 51 18 80 80 90.50% 10 − 41842122 41842184 63 16 49 18 8080 88.90% 5_GL000208v1_random − 10153 10215 63 16 49 18 80 80 85.50% 5 −47102206 47102267 62 16 49 18 80 80 88.90% 22 − 16039978 16040040 63 1649 20 80 80 90.20% 12 − 34833612 34833672 61 16 47 22 80 80 89.90% 5 −46417260 46417318 59 16 46 23 80 80 89.70% 5 − 46345090 46345147 58 1642 35 80 80 95.70% Un_KI270362v1 − 2841 2886 46 16 41 18 64 80 93.70% 3− 93710560 93710606 47 16 39 35 80 80 97.60% 5 − 50126203 50126419 21716 39 36 80 80 93.40% 5 − 50112923 50112967 45 16 38 35 80 80 91.40% 3 −93724825 93724870 46 16 37 34 80 80 89.40% 20 − 30166485 30166531 47 1637 34 80 80 89.40% 15 − 19788920 19788966 47 16 36 35 80 80 89.20%5_GL000208v1_random − 9643 9688 46 16 36 35 80 80 95.00% 20 − 3096741930967974 556 16 36 33 74 80 92.90% 10 − 41832639 41832680 42 16 34 35 8080 87.00% 2 − 94519233 94519278 46 16 28 18 47 80 96.70% 12 − 3483438134834410 30 16 27 53 80 80 100.00% 1 − 121857727 121858094 368 16 24 1847 80 90.00% 9 − 60574320 60574349 30 16 23 53 80 80 92.60% 1 −121975266 121975294 29 16 22 52 77 80 92.40% 12 − 34834689 34834714 2617 80 1 80 80 100.00% 3 + 91941787 91941866 80 17 80 1 80 80 100.00% 3 +91921547 91921626 80 17 80 1 80 80 100.00% 3 + 91910997 91911076 80 1780 1 80 80 100.00% 3 + 91853592 91853671 80 17 80 1 80 80 100.00% 3 +91807997 91808076 80 17 80 1 80 80 100.00% 3 + 91799327 91799406 80 1780 1 80 80 100.00% 3 + 91746937 91747016 80 17 80 1 80 80 100.00% 3 +91727032 91727111 80 17 80 1 80 80 100.00% 3 + 91703897 91703976 80 1780 1 80 80 100.00% 3 + 91694702 91694781 80 17 80 1 80 80 100.00% 3 +91683137 91683216 80 17 80 1 80 80 100.00% 3 + 91613047 91613126 80 1780 1 80 80 100.00% 3 + 91598592 91598671 80 17 80 1 80 80 100.00% 3 +91595362 91595441 80 17 80 1 80 80 100.00% 3 + 91573083 91573162 80 1780 1 80 80 100.00% 3 + 91559293 91559372 80 17 75 1 80 80 97.50% 3 −93711424 93711504 81 17 75 1 80 80 97.50% 3 − 93708539 93708619 81 17 751 80 80 97.50% 3 − 93705651 93705731 81 17 73 1 80 80 96.30% 3 −91545415 91545495 81 17 73 1 80 80 96.30% 3 − 91543203 91543283 81 17 721 80 80 96.30% 3 − 91547628 91548720 1093 17 71 1 80 80 95.00% 3 −93714472 93716075 1604 17 69 1 80 80 93.80% 3 − 91548640 91549739 110017 68 1 74 80 97.30% 16 − 38266052 38268848 2797 17 67 1 80 80 92.50% 5− 49631511 49631591 81 17 67 1 80 80 97.30% 3 − 91551530 91553302 177317 67 1 80 80 92.50% 12 − 37265483 37265563 81 17 67 1 80 80 92.50% 12 −34715472 34715887 416 17 67 1 80 80 92.50% 12 − 34832619 34832699 81 1767 1 80 80 92.50% 10 − 41839943 41840023 81 17 66 1 80 80 91.30% 10 −41841468 41841547 80 17 65 1 80 80 91.30% 5 − 49610156 49610576 421 1765 1 80 80 91.20% 5 − 49632528 49632608 81 17 64 1 80 80 91.10% 3 −91552546 91552626 81 17 63 1 80 80 90.00% 12 − 37238530 37238610 81 1762 1 80 80 91.90% 5 − 49625480 49625560 81 17 62 1 80 80 91.90% 16 −38276904 38276984 81 17 62 1 73 80 93.20% 16 − 38275892 38275965 74 1762 1 73 80 93.20% 16 − 38267074 38267147 74 17 61 1 74 80 91.90% 5 −49646145 49646219 75 17 61 1 74 80 91.90% 5 − 49644118 49644192 75 17 611 74 80 91.90% 5 − 49630838 49630912 75 17 61 1 74 80 91.90% 5 −49623787 49623861 75 17 61 1 74 80 91.90% 5 − 49617013 49617087 75 17 611 74 80 91.90% 5 − 49614973 49615047 75 17 61 1 74 80 91.90% 5 −49611520 49611594 75 17 61 1 80 80 88.80% 5 − 49610834 49610914 81 17 611 80 80 91.80% 16 − 38277919 38277999 81 17 61 1 74 80 91.90% 16 −38276231 38276305 75 17 61 1 80 80 88.80% 12 − 34829685 34829765 81 1761 1 80 80 88.80% 10 − 41841128 41841208 81 17 60 1 74 80 94.20%Un_KI270317v1 − 37196 37270 75 17 60 1 74 80 91.80% Un_KI270317v1 − 795869 75 17 60 1 80 80 90.60% 10 − 41839605 41839685 81 17 60 1 79 8088.70% 10 − 41833512 41833591 80 17 59 1 80 80 87.50% Un_KI270411v1 −1051 1131 81 17 59 1 74 80 90.60% Un_KI270304v1 − 723 797 75 17 59 1 7480 90.60% 5 − 49643782 49643856 75 17 59 1 80 80 87.50% 5 − 4962514049625220 81 17 59 1 74 80 90.60% 5 − 49622432 49622506 75 17 59 1 74 8090.60% 5 − 49615653 49615727 75 17 59 1 72 80 95.40% 3 − 9154354791547707 4161 17 59 1 72 80 91.70% 3 − 91544227 91544299 73 17 59 1 7480 90.60% 16 − 38276571 38276645 75 17 58 1 80 80 88.20% 10 − 4183215541837819 5665 17 57 1 72 80 90.30% 3 − 93706678 93706750 73 17 57 1 7480 89.20% 3 − 90499941 90500015 75 17 56 1 80 80 92.50% Un_KI270303v1 −808 888 81 17 56 1 71 80 90.20% 3 − 91546440 91546511 72 17 55 1 74 8087.90% 16 − 38279624 38279698 75 17 54 1 71 80 88.80% 1 − 122462723122462794 72 17 54 1 71 80 88.80% 1 − 122496505 122496576 72 17 52 1 7480 92.00% Un_KI270304v1 − 1403 1477 75 17 48 1 76 80 82.90% 7 − 6088458160884814 234 17 45 21 80 80 88.40% 4 − 67400112 67400172 61 17 45 1 8080 92.50% 16 − 38268768 38280038 11271 17 45 1 80 80 92.50% 16 −38268427 38268508 82 17 42 1 57 80 95.80% 3 − 91535155 91535725 571 1740 38 80 80 97.70% 12 − 37251765 37251808 44 17 40 38 80 80 97.70% 12 −34835050 34835093 44 17 36 32 74 80 93.10% 7 − 57889049 57889433 385 1734 43 80 80 94.80% 5 − 46416945 46416982 38 17 34 32 72 80 92.50% 3 −91549667 91551579 1913 17 34 32 72 80 91.70% 3 − 91551877 91551916 40 1734 43 80 80 94.80% 3 − 90490295 90490332 38 17 34 43 80 80 94.80% 12 −34716145 34716182 38 17 32 36 74 80 92.40% 7 − 57993147 57993186 40 1732 43 76 80 97.10% 11 − 55207299 55207332 34 17 30 43 74 80 96.90% 3 −90288971 90289002 32 17 30 43 74 80 96.90% 3 − 90635303 90635334 32 1730 43 74 80 96.90% 10 − 39361121 39361152 32 17 30 43 74 80 96.90% 10 −39361636 39361667 32 17 29 43 73 80 96.80% 3 − 93716002 93716032 31 1728 43 74 80 93.80% 3 − 90378865 90378896 32 17 27 46 72 80 100.00% 3 −91552893 91552919 27 17 26 43 74 80 90.70% 12 − 37356881 37356912 32 1726 43 74 80 90.70% 12 − 37675195 37675226 32 17 26 43 71 80 96.60% 10 −39353040 39353240 201 17 25 43 67 80 100.00% 16 − 38279971 38279995 2517 23 47 71 80 96.00% 10 − 41837748 41837772 25 17 22 51 72 80 100.00% 3− 91547972 91547993 22 18 75 4 80 80 98.80% 3 + 91673085 91673161 77 1875 4 80 80 98.80% 3 + 91631570 91631646 77 18 75 4 80 80 98.80% 3 +91628682 91628758 77 18 75 4 80 80 98.80% 3 + 91622897 91622973 77 18 754 80 80 98.80% 3 + 91617114 91617190 77 18 75 4 80 80 98.80% 3 +91611670 91611746 77 18 75 4 80 80 98.80% 3 + 91608778 91608854 77 18 754 80 80 98.80% 3 + 91605888 91605964 77 18 75 4 80 80 98.80% 3 +91600103 91600179 77 18 75 4 80 80 98.80% 3 + 91597212 91597288 77 18 754 80 80 98.80% 3 + 91593982 91594058 77 18 75 4 80 80 98.80% 3 +91591090 91591166 77 18 75 4 80 80 98.80% 3 + 91589047 91589123 77 18 754 80 80 98.80% 3 + 91574595 91574671 77 18 75 4 80 80 98.80% 3 +91569150 91569226 77 18 75 4 80 80 98.80% 3 + 91566255 91566331 77 18 734 80 80 97.50% 3 − 91552909 91552985 77 18 73 4 80 80 97.50% 3 −91550022 91550098 77 18 70 1 80 80 95.00% 3 − 93713821 93713902 82 18 644 80 80 92.30% 3 − 93710593 93712882 2290 18 64 4 80 80 92.30% 3 −93707708 93709992 2285 18 60 4 80 80 89.70% 3 − 91540858 91541781 924 1860 4 80 80 89.70% 3 − 91546798 91546876 79 18 58 4 80 80 88.40% 3 −93713482 93713560 79 18 58 4 80 80 88.40% 3 − 91550699 91550777 79 18 584 80 80 88.40% 3 − 91544583 91544661 79 18 55 14 80 80 92.60% 5 −49624825 49632618 7794 18 55 1 74 80 87.90% 12 − 34832649 34833232 58418 53 7 80 80 86.50% 16 − 38267091 38267166 76 18 53 13 80 80 89.80% 12− 37258671 37267612 8942 18 52 13 80 80 88.30% 3 − 91549683 91549750 6818 50 4 53 80 100.00% 3 − 93707060 93707109 50 18 46 4 80 80 80.60% 3 −90626989 90643226 16238 18 46 15 80 80 84.90% 12 − 37264491 37264556 6618 43 4 53 80 94.00% 12 − 34717885 34718104 220 18 41 7 80 80 78.40% 3 −91286126 91286200 75 18 40 4 80 80 76.70% Un_KI270512v1 − 17870 17948 7918 40 4 80 80 76.70% Un_KI270442v1 − 207790 207868 79 18 39 7 53 8093.50% 12 − 37257287 37257391 105 18 38 7 53 80 91.50% Un_KI270317v1 −37241 37289 49 18 38 7 53 80 91.50% 5 − 49611225 49611273 49 18 38 13 8080 86.60% 3 − 90545653 90643215 97563 18 38 7 53 80 91.50% 16 − 3828034338280391 49 18 38 4 80 80 75.40% 11 − 50779589 50779667 79 18 36 7 53 8089.40% Un_KI270317v1 − 1519 1567 49 18 33 4 80 80 97.30% 7 − 6221151462211593 80 18 33 13 53 80 90.30% 3 − 93712494 93712534 41 18 33 13 5380 97.20% 16 − 38266438 38266818 381 18 33 13 53 80 90.30% 16 − 3826881938268859 41 18 33 13 53 80 90.30% 12 − 34715523 34715563 41 18 31 13 5180 89.80% 4 − 67399485 67399523 39 18 31 15 53 80 89.80% 12 − 3483301034833048 39 18 29 13 51 80 87.20% 16 − 38277291 38277329 39 18 25 2 2780 100.00% 17 − 22665014 22665041 28 18 23 4 27 80 100.00% 3 − 9370861693708641 26 18 23 4 27 80 100.00% 3 − 93711501 93711526 26 18 23 7 30 80100.00% 10 − 41840863 41840888 26 18 20 59 80 80 95.50% Un_KI270579v1 −29179 29200 22 18 20 59 80 80 95.50% 9 − 63079723 63079744 22 18 20 5980 80 95.50% 21 − 5286988 5287009 22 18 20 47 80 80 79.50% 20 − 2973339629733429 34 2 80 1 80 80 100.00% 3 + 91813172 91813251 80 2 80 1 80 80100.00% 3 + 91807388 91807467 80 2 80 1 80 80 100.00% 3 + 9177524791775326 80 2 80 1 80 80 100.00% 3 + 91755003 91755082 80 2 80 1 80 80100.00% 3 + 91743776 91743855 80 2 80 1 80 80 100.00% 3 + 9172063891720717 80 2 80 1 80 80 100.00% 3 + 91698522 91698601 80 2 80 1 80 80100.00% 3 + 91688311 91688390 80 2 80 1 80 80 100.00% 3 + 9166807391668152 80 2 80 1 80 80 100.00% 3 + 91665182 91665261 80 2 80 1 80 80100.00% 3 + 91651048 91651127 80 2 80 1 80 80 100.00% 3 + 9164526691645345 80 2 80 1 80 80 100.00% 3 + 91642373 91642452 80 2 80 1 80 80100.00% 3 + 91626562 91626641 80 2 80 1 80 80 100.00% 3 + 9159475391594832 80 2 80 1 80 80 100.00% 3 + 91591862 91591941 80 2 78 1 80 8098.80% 3 − 93706258 93706337 80 2 78 1 80 80 98.80% 3 − 9371203193712110 80 2 78 1 80 80 98.80% 3 − 91543807 91543886 80 2 78 1 80 8098.80% 3 − 91549247 91549326 80 2 76 1 80 80 97.50% 3 − 9370914693709225 80 2 76 1 80 80 97.50% 3 − 91548231 91548310 80 2 76 1 80 8097.50% 3 − 91552136 91552215 80 2 72 1 79 80 92.30% 3 − 9154602591546101 77 2 68 1 80 80 92.50% 12 − 37263720 37263799 80 2 68 1 80 8092.50% 12 − 34718442 34718521 80 2 66 1 80 80 91.30% 3 − 9370846893708547 80 2 66 1 80 80 91.30% 3 − 93711353 93711432 80 2 66 1 80 8091.30% 12 − 37261003 37261082 80 2 66 1 80 80 91.30% 10 − 4183953441839613 80 2 64 1 72 80 94.50% 3 − 90600358 90600429 72 2 64 1 80 8090.00% 12 − 37238799 37238878 80 2 64 1 80 80 90.00% 12 − 3725169437251773 80 2 64 1 80 80 90.00% 12 − 37259991 37260070 80 2 64 1 80 8090.00% 12 − 37260331 37260410 80 2 64 1 80 80 90.00% 12 − 3483497934835058 80 2 64 1 80 80 90.00% 10 − 41840718 41840797 80 2 63 1 80 8090.00% 3 − 93706938 93707186 249 2 63 1 72 80 94.50% 3 − 9055137790552643 1267 2 62 1 79 80 89.90% 3 − 90692725 90702557 9833 2 62 1 7280 93.10% 3 − 90719111 90719182 72 2 62 1 72 80 93.10% 3 − 9052037890520449 72 2 62 1 72 80 93.10% 3 − 90500383 90500454 72 2 62 1 80 8088.80% 12 − 37239815 37239894 80 2 62 1 72 80 93.10% 12 − 3725379737253868 72 2 62 1 72 80 93.10% 12 − 34833574 34833645 72 2 62 1 80 8088.80% 10 − 41833440 41833519 80 2 62 1 72 80 93.10% 10 − 4183090441830975 72 2 61 1 72 80 93.10% 3 − 90408885 90686071 277187 2 60 1 7280 91.70% 3 − 90581736 90581807 72 2 60 1 72 80 91.70% 12 − 3471608234716153 72 2 60 2 69 80 94.20% 12 − 34821492 34821559 68 2 60 1 72 8091.70% 12 − 34833234 34833305 72 2 59 1 72 80 91.60% 1 − 124961063124961305 243 2 59 1 72 80 91.70% 1 − 124988057 124988299 243 2 58 1 7280 90.30% Un_KI270303v1 − 1085 1156 72 2 58 1 80 80 86.30% 3 − 9155315291553231 80 2 58 1 72 80 90.30% 3 − 90548114 90548185 72 2 56 7 72 8092.50% Un_KI270366v1 − 2285 2350 66 2 56 1 72 80 88.90%5_GL000208v1_random − 43187 43258 72 2 56 1 72 80 88.90% 3 − 9057816790578238 72 2 56 1 66 80 92.50% 3 − 90488191 90488256 66 2 56 1 70 8093.80% 12 − 34716424 34716494 71 2 56 1 66 80 92.50% 12 − 3482047734820542 66 2 56 1 66 80 92.50% 12 − 34820816 34820881 66 2 55 1 65 8092.40% 3 − 90690836 90690900 65 2 54 1 72 80 87.50% 5 − 4961388549613956 72 2 54 1 72 80 87.50% 3 − 90616783 90616854 72 2 54 1 72 8087.50% 10 − 41839202 41839273 72 2 53 1 63 80 92.10% 12 − 3725210737252169 63 2 53 1 65 80 90.80% 12 − 34710310 34710374 65 2 52 7 68 8092.00% 5_GL000208v1_random − 5353 5414 62 2 52 1 66 80 89.40% 3 −91530844 91530909 66 2 52 1 66 80 89.40% 3 − 90502459 90502524 66 2 52 766 80 93.40% 20 − 30163565 30163624 60 2 52 1 72 80 86.20% 1 − 124959871124959942 72 2 50 1 72 80 84.80% 3 − 90687654 90687725 72 2 50 1 66 8087.90% 3 − 90511948 90512013 66 2 50 1 66 80 87.90% 10 − 4183260141832666 66 2 50 1 72 80 84.80% 1 − 121923711 121923782 72 2 49 8 66 8091.60% 12 − 34711333 34711391 59 2 48 7 64 80 91.40% 5 − 4633334246333399 58 2 48 17 72 80 92.90% 12 − 37266439 37266494 56 2 46 17 69 8094.40% 10 − 39190659 39353539 162881 2 45 17 69 80 92.50% 11 − 5493232454932376 53 2 42 22 69 80 93.80% 11 − 54877517 54877564 48 2 42 17 66 8092.00% 10 − 39289661 39289710 50 2 41 23 69 80 93.70% 11 − 5488163254881678 47 2 41 17 65 80 91.90% 11 − 50546179 50546227 49 2 41 22 69 8093.70% 10 − 39353659 39363160 9502 2 40 21 68 80 91.70%16_KI270728v1_random − 612954 613001 48 2 38 30 72 80 95.40%Un_KI270317v1 − 1065 1109 45 2 38 23 66 80 93.20% 11 − 55230762 5523080544 2 37 17 63 80 89.40% 11 − 55066450 55066496 47 2 36 33 72 80 95.00% 5− 49612186 49612225 40 2 36 22 69 80 87.50% 11 − 48706435 48706482 48 235 1 41 80 92.70% 10 − 41828481 41828521 41 2 33 22 72 80 82.40% 1 −121975741 121975791 51 2 28 1 30 80 96.70% 22 − 15996865 15996894 30 225 1 27 80 96.30% X − 62524981 62525007 27 2 23 1 27 80 92.60%Un_KI270411v1 − 1033 1059 27 2 22 47 72 80 92.40% 22 − 15997164 1599718926 2 20 32 51 80 100.00% X − 62525290 62525309 20 2 20 22 41 80 100.00%11 − 50517626 50517645 20 3 80 1 80 80 100.00% 3 + 92019076 92019155 803 80 1 80 80 100.00% 3 + 92013291 92013370 80 3 80 1 80 80 100.00% 3 +91944231 91944310 80 3 80 1 80 80 100.00% 3 + 91923991 91924070 80 3 801 80 80 100.00% 3 + 91893016 91893095 80 3 80 1 80 80 100.00% 3 +91878476 91878555 80 3 80 1 80 80 100.00% 3 + 91844466 91844545 80 3 801 80 80 100.00% 3 + 91833236 91833315 80 3 80 1 80 80 100.00% 3 +91827791 91827870 80 3 80 1 80 80 100.00% 3 + 91819116 91819195 80 3 801 80 80 100.00% 3 + 91743936 91744015 80 3 80 1 80 80 100.00% 3 +91688471 91688550 80 3 80 1 80 80 100.00% 3 + 91679801 91679880 80 3 801 80 80 100.00% 3 + 91645426 91645505 80 3 80 1 80 80 100.00% 3 +91632501 91632580 80 3 80 1 80 80 100.00% 3 + 91623831 91623910 80 3 781 80 80 98.80% 3 − 91549087 91549166 80 3 78 1 80 80 98.80% 3 − 9154364791543726 80 3 76 1 80 80 97.50% 3 − 93711871 93711950 80 3 76 1 80 8097.50% 3 − 93708986 93709065 80 3 72 1 80 80 95.00% 3 − 9370609893706177 80 3 70 1 80 80 93.80% 3 − 91545864 91545943 80 3 68 1 80 8092.50% 3 − 91548072 91548151 80 3 66 1 80 80 93.10% 3 − 9155265691552734 79 3 66 1 80 80 91.30% 3 − 91551977 91552056 80 3 65 1 80 8093.00% Un_KI270412v1 − 364 442 79 3 65 1 80 80 95.80% 12 − 3725915337267369 8217 3 65 1 80 80 93.00% 12 − 37264917 37264995 79 3 65 1 80 8093.00% 12 − 34715915 34715993 79 3 64 1 80 80 91.70% 3 − 9370966393709741 79 3 64 1 80 80 91.70% 3 − 93706780 93706858 79 3 63 1 80 8094.40% Un_KI270411v1 − 1161 1579 419 3 63 1 80 80 94.40% 3 − 9370864993710078 1430 3 63 1 80 80 94.40% 3 − 91540944 91542200 1257 3 63 1 8080 90.20% 3 − 91540605 91540682 78 3 63 1 80 80 91.60% 12 − 3726321337263291 79 3 63 1 80 80 91.60% 12 − 37261524 37261602 79 3 63 1 80 8091.60% 12 − 37260172 37260250 79 3 63 1 80 80 91.60% 12 − 3725227137252349 79 3 63 1 80 80 90.30% 12 − 34719301 34719379 79 3 63 1 80 8091.60% 12 − 34717608 34717686 79 3 63 1 80 80 91.60% 10 − 4184157741841655 79 3 63 1 80 80 91.60% 10 − 41840899 41840977 79 3 63 1 80 8091.60% 10 − 41839713 41839791 79 3 62 1 80 80 94.40% 3 − 9371153493715508 3975 3 62 1 79 80 94.30% 3 − 93715432 93715839 408 3 62 1 80 8090.30% 3 − 93714245 93714323 79 3 62 5 80 80 92.20% 3 − 9062297190624509 1539 3 62 1 80 80 94.30% 12 − 37248829 37262620 13792 3 62 1 8080 91.50% 12 − 37266951 37267029 79 3 62 1 80 80 94.40% 10 − 4184005341840297 245 3 61 1 78 80 94.30% 4 − 67400224 67400640 417 3 61 1 80 8091.40% 3 − 93716104 93716182 79 3 61 1 80 80 90.20% 12 − 3726661137266689 79 3 61 1 80 80 90.20% 10 − 41837849 41837927 79 3 60 1 80 8093.00% 12 − 37263798 37263978 181 3 60 1 80 80 90.00% 12 − 3726084437260922 79 3 60 1 80 80 87.50% 12 − 37252949 37253028 80 3 60 1 80 8090.00% 12 − 34716596 34716674 79 3 59 1 76 80 91.10% Un_KI270411v1 − 150224 75 3 59 1 80 80 88.80% 12 − 37263561 37263639 79 3 59 1 80 80 91.60%12 − 34716936 34719041 2106 3 58 1 80 80 87.50% 3 − 91544329 91544407 793 57 1 78 80 88.50% 12 − 37264240 37264316 77 3 56 8 80 80 90.70% 12 −37265593 37265664 72 3 55 8 80 80 87.70% 3 − 91545525 91545597 73 3 55 180 80 88.10% 16 − 38280068 38280146 79 3 54 8 80 80 87.50% 3 − 9370576193705831 71 3 54 1 80 80 87.90% 12 − 37259832 37259910 79 3 52 1 76 8084.90% Un_KI270412v1 − 708 781 74 3 52 6 71 80 87.50% 3 − 9069152090691584 65 3 52 1 80 80 85.30% 16 − 38268538 38268616 79 3 52 8 80 8090.00% 12 − 34831491 34831562 72 3 51 1 80 80 94.40% 4 − 6739954267399620 79 3 51 5 75 80 94.80% 3 − 90534315 90581982 47668 3 50 1 80 8081.30% 10 − 41842256 41842335 80 3 49 1 80 80 94.70% 5 − 4635618746356709 523 3 49 2 80 80 89.50% 16 − 38265816 38265893 78 3 48 5 75 8092.40% 3 − 90514274 90514343 70 3 48 2 80 80 89.30% 16 − 3827599538276072 78 3 46 5 80 80 94.40% Un_KI270538v1 − 57928 58275 348 3 45 578 80 90.20% 21 − 7246234 7246306 73 3 45 5 78 80 90.20% 21 − 53162155316287 73 3 44 5 75 80 91.70% 3 − 90634901 90634970 70 3 44 5 75 8091.70% 3 − 90510249 90510318 70 3 42 1 80 80 93.20% Un_KI270412v1 − 10421120 79 3 42 1 76 80 91.40% Un_KI270412v1 − 28 102 75 3 41 8 75 8091.20% 3 − 90609496 90609562 67 3 40 5 80 80 97.70% 3 − 9058180690623148 41343 3 36 5 75 80 92.20% 3 − 90630314 90630383 70 3 36 5 75 8092.20% 3 − 90458369 90458438 70 3 36 5 75 80 92.20% 3 − 9042817690428245 70 3 33 44 80 80 94.60% 3 − 91546541 91546577 37 3 32 1 34 8097.10% Un_KI270411v1 − 1884 1917 34 3 32 1 34 80 97.10% 4 − 6740026767400300 34 3 31 1 35 80 94.30% 3 − 91546585 91546619 35 3 31 2 34 8097.00% 16 − 38267222 38267254 33 3 30 1 34 80 94.20% Un_KI270411v1 −1206 1239 34 3 29 1 33 80 94.00% Un_KI270411v1 − 2563 2595 33 3 29 45 7980 91.50% 3 − 93710001 93710035 35 3 29 44 80 80 89.20% 3 − 9371576193715797 37 3 29 5 34 80 100.00% 12 − 34716981 34717175 195 3 28 1 34 8091.20% 3 − 91540989 91541022 34 3 26 45 80 80 86.20% Un_KI270411v1 −1501 1536 36 3 25 1 27 80 96.30% Un_KI270411v1 − 874 900 27 3 23 44 7080 92.60% Un_KI270411v1 − 1849 1875 27 3 23 58 80 80 100.00%Un_KI270411v1 − 2518 2540 23 3 23 8 34 80 92.60% 3 − 93708693 9370871927 3 23 54 80 80 92.60% 12 − 34718963 34718989 27 4 80 1 80 80 100.00%3 + 91606900 91606979 80 4 80 1 80 80 100.00% 3 + 91604008 91604087 80 480 1 80 80 100.00% 3 + 91601115 91601194 80 4 80 1 80 80 100.00% 3 +91598224 91598303 80 4 80 1 80 80 100.00% 3 + 91594993 91595072 80 4 801 80 80 100.00% 3 + 91592102 91592181 80 4 80 1 80 80 100.00% 3 +91590059 91590138 80 4 80 1 80 80 100.00% 3 + 91584276 91584355 80 4 801 80 80 100.00% 3 + 91581383 91581462 80 4 80 1 80 80 100.00% 3 +91575603 91575682 80 4 80 1 80 80 100.00% 3 + 91572715 91572794 80 4 801 80 80 100.00% 3 + 91567270 91567349 80 4 80 1 80 80 100.00% 3 +91564374 91564453 80 4 80 1 80 80 100.00% 3 + 91561820 91561899 80 4 801 80 80 100.00% 3 + 91558925 91559004 80 4 80 1 80 80 100.00% 3 +91556038 91556117 80 4 78 1 80 80 96.30% 3 + 93062400 93062478 79 4 76 180 80 97.50% 3 − 91551560 91551639 80 4 76 1 80 80 97.50% 3 − 9154867091548749 80 4 74 1 80 80 96.30% 3 − 91552576 91552655 80 4 70 1 80 8093.80% 3 − 91545445 91545524 80 4 66 1 80 80 91.30% 3 − 9154323391543312 80 4 62 1 80 80 84.00% 3 − 93711459 93711533 75 4 62 1 80 8084.00% 3 − 93708574 93708648 75 4 62 1 80 80 84.00% 3 − 9370568693705760 75 4 61 1 80 80 88.70% 12 − 34832649 34832728 80 4 60 1 80 8087.90% 3 − 91547658 91547736 79 4 59 1 80 80 87.50% 12 − 3725833737258812 476 4 59 1 80 80 87.50% 10 − 41839973 41841576 1604 4 58 1 8080 86.30% 3 − 93715351 93715430 80 4 58 1 80 80 86.30% 16 − 3826709738267176 80 4 57 1 80 80 86.30% 5 − 49624151 49630941 6791 4 56 8 80 8089.10% 12 − 37263481 37263722 242 4 55 8 80 80 87.70% 3 − 9371568193715753 73 4 55 1 80 80 85.00% 16 − 38278968 38280401 1434 4 51 1 80 8082.50% 5 − 49643806 49644221 416 4 51 1 77 80 83.20% 3 − 9370602193706097 77 4 51 8 80 80 85.00% 3 − 90360501 90360573 73 4 50 1 80 8081.30% 5 − 49631882 49631961 80 4 50 11 80 80 91.10% 3 − 9155036691550434 69 4 50 1 80 80 81.30% 16 − 38279988 38280067 80 4 49 8 80 8083.60% 9 − 63075806 63075878 73 4 49 8 80 80 83.60% 7 − 5791327257913344 73 4 49 8 80 80 83.60% 21 − 12983436 12983508 73 4 49 14 80 8086.60% 12 − 37259752 37259818 67 4 47 8 80 80 82.20% 7 − 5796764557967717 73 4 46 8 80 80 91.10% 3 − 93779766 93780178 413 4 46 17 80 8092.00% 3 − 91553253 91553315 63 4 44 11 80 80 81.50% 3 − 9152582691525895 70 4 44 8 80 80 80.60% 15 − 17076410 17076482 73 4 44 12 77 8083.40% 12 − 37254629 37254694 66 4 42 11 80 80 80.00% 3 − 9153916191539230 70 4 39 1 42 80 97.70% 22 − 15156952 15157116 165 4 38 1 42 8095.30% 5 − 49615035 49615076 42 4 38 1 42 80 95.30% 5 − 4961707549617116 42 4 38 1 42 80 95.30% 2 − 94513488 94513529 42 4 38 1 42 8095.30% 12 − 34834101 34834142 42 4 38 1 42 80 95.30% 10 − 4184119641841237 42 4 37 1 42 80 95.20% 22 − 15155755 15155967 213 4 36 1 42 8092.90% 5 − 49612996 49613037 42 4 36 1 42 80 92.90% 16 − 3827832638278367 42 4 35 6 42 80 97.30% 2 − 94521706 94521742 37 4 34 11 80 8087.50% 16 − 34612243 34612311 69 4 34 8 42 80 100.00% 10 − 4183967341839874 202 4 33 8 40 80 100.00% Un_KI270329v1 − 476 508 33 4 33 8 4480 94.60% 7 − 57981340 57981376 37 4 33 8 40 80 100.00% 3 − 9064319490643226 33 4 33 8 42 80 97.20% 3 − 90485770 90485804 35 4 33 6 42 8094.60% 22 − 15163435 15163471 37 4 33 8 42 80 97.20% 20 − 2888889728888931 35 4 33 8 69 80 89.20% 16 − 35936044 35936104 61 4 33 8 69 8094.60% 16 − 35940461 35940523 63 4 33 8 42 80 97.20% 15 − 1978237619782410 35 4 33 6 42 80 94.60% 14 − 18233662 18233698 37 4 32 11 42 80100.00% Un_KI270411v1 − 780 811 32 4 32 11 42 80 100.00% Un_KI270304v1 −785 816 32 4 32 11 42 80 100.00% 5 − 49645870 49645901 32 4 32 11 42 80100.00% 5 − 49633616 49633647 32 4 32 8 42 80 97.20% 16 − 3826815538268358 204 4 32 11 42 80 100.00% 16 − 38278666 38278697 32 4 32 11 4280 100.00% 15 − 19786460 19786491 32 4 31 8 42 80 94.30% Un_KI270591v1 −4770 4804 35 4 31 11 41 80 100.00% Un_KI270310v1 − 1066 1096 31 4 31 875 80 68.20% 16_KI270728v1_random − 611681 611746 66 4 30 8 69 80 96.90%7 − 61845779 61845841 63 4 30 11 42 80 96.90% 5 − 50198435 50198466 32 430 11 42 80 96.90% 3 − 90487115 90487146 32 4 30 11 42 80 96.90% 3 −90489170 90489201 32 4 30 11 42 80 96.90% 2 − 94538605 94538636 32 4 3011 42 80 96.90% 15 − 19784590 19784621 32 4 29 8 38 80 96.80% 22 −16077061 16077091 31 4 28 8 68 80 96.70% Y − 17673610 17673671 62 4 28 968 80 96.70% 9 − 64213671 64213731 61 4 28 6 33 80 100.00% 3 − 9371353593713562 28 4 28 11 42 80 93.80% 3 − 90489510 90489541 32 4 28 15 42 80100.00% 11 − 48936956 48936983 28 4 26 11 40 80 93.40% X − 6252557262525601 30 4 26 11 42 80 90.70% Un_KI270366v1 − 4799 4830 32 4 26 13 4280 93.40% 5_GL000208v1_random − 73822 73851 30 4 26 11 42 80 90.70% 2 −94514039 94514070 32 4 26 11 42 80 90.70% 11 − 55259545 55259576 32 4 2057 80 80 91.70% 4 − 67398783 67398806 24 5 80 1 80 80 100.00% 3 +91850412 91850491 80 5 80 1 80 80 100.00% 3 + 91838842 91838921 80 5 801 80 80 100.00% 3 + 91822167 91822246 80 5 80 1 80 80 100.00% 3 +91813492 91813571 80 5 80 1 80 80 100.00% 3 + 91781352 91781431 80 5 801 80 80 100.00% 3 + 91775567 91775646 80 5 80 1 80 80 100.00% 3 +91752432 91752511 80 5 80 1 80 80 100.00% 3 + 91709392 91709471 80 5 801 80 80 100.00% 3 + 91698842 91698921 80 5 80 1 80 80 100.00% 3 +91626882 91626961 80 5 80 1 80 80 100.00% 3 + 91618207 91618286 80 5 801 80 80 100.00% 3 + 91595412 91595491 80 5 80 1 80 80 100.00% 3 +91595073 91595152 80 5 80 1 80 80 100.00% 3 + 91592182 91592261 80 5 801 80 80 100.00% 3 + 91581463 91581542 80 5 80 1 80 80 100.00% 3 +91556118 91556197 80 5 76 1 80 80 97.50% 3 − 91548929 91549008 80 5 76 180 80 97.50% 3 − 91551819 91551898 80 5 76 1 80 80 97.50% 3 − 9154348991543568 80 5 75 1 80 80 97.50% 3 − 93711713 93712131 419 5 74 1 80 8096.30% 3 − 91547914 91547993 80 5 72 1 80 80 95.00% 3 − 9370594093706019 80 5 72 1 80 80 95.00% 3 − 93708828 93708907 80 5 71 1 80 8095.00% 5 − 49646260 49646508 249 5 71 1 80 80 95.00% 10 − 4183972441839972 249 5 70 1 80 80 93.80% 3 − 91545706 91545785 80 5 70 1 80 8093.80% 3 − 90507030 90507109 80 5 70 1 80 80 93.80% 10 − 4184141841841497 80 5 67 1 80 80 94.70% 3 − 93708317 93708568 252 5 67 1 80 8094.70% 3 − 93711202 93711453 252 5 67 1 80 80 92.50% 3 − 9154740891547657 250 5 67 1 74 80 91.70% 3 − 91543161 91543232 72 5 66 1 78 8092.40% X − 62516732 62516809 78 5 66 1 80 80 91.30% 22 − 1519044515190524 80 5 66 1 80 80 93.50% 2 − 94517164 94518262 1099 5 66 1 80 8091.30% 2 − 94519378 94519457 80 5 66 1 80 80 91.30% 2 − 9451494994515028 80 5 66 1 74 80 96.00% 16 − 38276012 38276254 243 5 66 1 80 8091.30% 14 − 18259650 18259729 80 5 66 1 74 80 94.60% 12 − 3726543937265512 74 5 65 1 80 80 93.40% 5 − 49610955 49611203 249 5 65 1 80 8093.40% 16 − 38277699 38277948 250 5 65 1 79 80 91.20% 1 − 124978521124978599 79 5 64 1 80 80 90.00% 3 − 90492064 90492143 80 5 64 1 80 8087.40% 22_KI270738v1_random − 88645 88723 79 5 64 1 80 80 90.00% 20 −29700375 29700454 80 5 64 1 77 80 92.30% 12 − 37344561 37373656 29096 564 3 74 80 94.50% 12 − 37255224 37255295 72 5 64 1 74 80 93.30% 10 −41832111 41832184 74 5 64 1 74 80 93.30% 10 − 41839561 41839634 74 5 631 79 80 89.90% 7 − 62557464 62557542 79 5 63 1 77 80 91.00% 3 − 9071438190714457 77 5 63 1 80 80 89.90% 22 − 16030865 16031285 421 5 63 1 80 8092.00% 22 − 15158885 15172104 13220 5 63 1 80 80 92.20% 2 − 9450978794510033 247 5 63 1 73 80 93.20% 12 − 37248335 37248407 73 5 62 1 80 8088.80% X − 62515203 62515282 80 5 62 1 80 80 88.80% Un_KI270538v1 −63013 63092 80 5 62 1 74 80 91.90% 5 − 49626456 49626529 74 5 62 1 80 8088.80% 22 − 15195900 15195979 80 5 62 1 80 80 93.10% 20 − 3016646030166881 422 5 62 1 80 80 88.80% 19 − 27403689 27403768 80 5 62 1 80 8088.80% 16_KI270728v1_random − 1758141 1758220 80 5 62 1 74 80 91.90% 16− 38279574 38279647 74 5 62 1 74 80 91.90% 12 − 37261030 37261103 74 562 1 80 80 88.80% 12 − 37257918 37257997 80 5 62 1 74 80 91.90% 12 −34715763 34715836 74 5 62 1 80 80 88.80% 11 − 50722395 50722474 80 5 621 80 80 88.80% 11 − 48915459 48915538 80 5 61 1 80 80 89.70% 2 −94515802 94516052 251 5 60 11 80 80 92.90% 5_GL000208v1_random − 7898579054 70 5 60 11 80 80 92.90% 5_GL000208v1_random − 81199 81268 70 5 601 74 80 90.60% 3 − 91549954 91550027 74 5 60 1 80 80 87.50% 22 −15190790 15190869 80 5 60 1 80 80 87.50% 20 − 30160704 30160783 80 5 601 74 80 90.60% 16 − 38279914 38279987 74 5 60 1 80 80 87.50% 14 −18259995 18260074 80 5 59 1 75 80 89.40% 20 − 30994129 30994203 75 5 599 80 80 94.10% 16 − 38279059 38279299 241 5 59 1 73 80 90.50% 12 −37263060 37263132 73 5 59 10 76 80 94.10% 12 − 37363722 37363788 67 5 5910 74 80 97.00% 1 − 124985180 124985415 236 5 58 13 80 80 92.70%Un_KI270538v1 − 57360 57427 68 5 58 1 80 80 90.30% 5 − 50137417 50137667251 5 58 1 80 80 90.30% 5 − 50139972 50140222 251 5 58 1 80 80 86.30% 3− 90695300 90695379 80 5 58 1 80 80 86.30% 22 − 15177819 15177898 80 558 1 80 80 86.30% 20 − 30970113 30970192 80 5 58 1 74 80 89.20% 12 −34717454 34717527 74 5 57 1 80 80 86.30% 14 − 18235244 18243179 7936 557 13 77 80 93.90% 12 − 37359727 37359791 65 5 56 1 80 80 87.90% 9 −60554373 60554623 251 5 56 1 80 80 85.00% 21 − 12978419 12978498 80 5 561 77 80 87.10% 14 − 18227405 18233963 6559 5 55 14 80 80 91.10%5_GL000208v1_random − 86650 86716 67 5 55 10 80 80 95.10% 12 − 3734065337340894 242 5 53 10 80 80 87.40% 14 − 18262720 18262790 71 5 53 1 71 8089.60% 11 − 55255346 55255572 227 5 50 1 80 80 90.40% 2 − 9453439594534474 80 5 47 9 80 80 84.10% 22 − 15176456 15176696 241 5 47 28 80 8094.40% 12 − 37373414 37373466 53 5 45 28 80 80 92.50% Un_KI270522v1 −3242 3294 53 5 43 10 56 80 95.80% 1 − 124994759 124994805 47 5 42 1 5280 90.40% 22 − 15159083 15159134 52 5 38 13 56 80 93.20% 15 − 1979488319794926 44 5 36 1 48 80 87.50% 2 − 94525362 94525409 48 5 35 2 40 8094.90% 14 − 18227613 18227651 39 5 28 10 41 80 93.80% 2 − 9451719994517230 32 5 27 48 80 80 91.00% 14 − 18243101 18243133 33 6 80 1 80 80100.00% 3 + 91856276 91856355 80 6 80 1 80 80 100.00% 3 + 9183058591830664 80 6 80 1 80 80 100.00% 3 + 91787215 91787294 80 6 80 1 80 80100.00% 3 + 91749620 91749699 80 6 80 1 80 80 100.00% 3 + 9173839691738475 80 6 80 1 80 80 100.00% 3 + 91729715 91729794 80 6 80 1 80 80100.00% 3 + 91721041 91721120 80 6 80 1 80 80 100.00% 3 + 9170658091706659 80 6 80 1 80 80 100.00% 3 + 91688711 91688790 80 6 80 1 80 80100.00% 3 + 91685820 91685899 80 6 80 1 80 80 100.00% 3 + 9167136691671445 80 6 80 1 80 80 100.00% 3 + 91662690 91662769 80 6 80 1 80 80100.00% 3 + 91651450 91651529 80 6 80 1 80 80 100.00% 3 + 9162407191624150 80 6 80 1 80 80 100.00% 3 + 91601275 91601354 80 6 80 1 80 80100.00% 3 + 91572875 91572954 80 6 78 1 80 80 98.80% 3 − 9370586093705939 80 6 78 1 80 80 98.80% 3 − 93708748 93708827 80 6 78 1 80 8098.80% 3 − 93711633 93711712 80 6 73 1 80 80 96.30% 3 − 9154783391547913 81 6 72 1 80 80 95.00% 3 − 91548849 91548928 80 6 70 1 80 8093.80% 3 − 91545626 91545705 80 6 68 1 80 80 92.50% 3 − 9155173991551818 80 6 63 1 80 80 90.00% 12 − 37265353 37265643 291 6 63 1 80 8090.00% 10 − 41839475 41839763 289 6 61 1 80 80 88.80% 3 − 9370840993708698 290 6 61 1 80 80 88.80% 3 − 93711294 93711583 290 6 61 1 80 8088.80% 3 − 91545285 91545576 292 6 61 1 80 80 88.80% 3 − 9154750091547786 287 6 61 1 80 80 88.80% 12 − 34716696 34716986 291 6 60 1 80 8087.50% 12 − 37251635 37251714 80 6 60 1 80 80 87.50% 12 − 3483492034834999 80 6 59 1 80 80 87.50% 12 − 34718383 34718674 292 6 59 1 80 8087.50% 10 − 41839813 41840103 291 6 59 8 80 80 90.50% 10 − 4184133841841410 73 6 58 1 80 80 86.30% Un_KI270411v1 − 1939 2018 80 6 58 1 8080 86.30% 3 − 91550206 91550285 80 6 58 1 80 80 86.30% 12 − 3725305037253129 80 6 57 1 80 80 86.30% 3 − 91543075 91543362 288 6 57 1 80 8086.30% 12 − 37254466 37254756 291 6 53 8 80 80 86.40% 12 − 3726230337262375 73 6 53 12 80 80 88.50% 12 − 37249945 37250013 69 6 53 12 80 8088.50% 12 − 37247568 37247636 69 6 52 13 80 80 88.30% 16 − 3827711438277181 68 6 51 12 80 80 87.00% 12 − 37247908 37247976 69 6 51 12 80 8087.00% 12 − 37254126 37254194 69 6 49 12 80 80 85.60% 12 − 3483023434830302 69 6 48 9 80 80 83.40% 3 − 91518385 91518456 72 6 47 1 80 8080.00% 2 − 94532265 94532384 120 6 46 9 80 80 82.00% 3 − 9151989591519966 72 6 45 4 80 80 76.40% 12 − 37639835 37639910 76 6 44 12 75 8084.40% Y − 10231696 10231759 64 6 44 12 75 80 84.40% 5 − 5022490650224969 64 6 44 11 80 80 81.50% 11 − 55259007 55259076 70 6 43 12 80 8092.20% 12 − 37240096 37240504 409 6 42 12 75 80 82.90% 11 − 5071170150711764 64 6 42 9 80 80 87.50% 11 − 48938814 48939056 243 6 41 12 75 8091.90% 11 − 48795804 48796209 406 6 39 13 75 80 81.00% 16 − 3420129634201358 63 6 36 14 75 80 86.40% 3 − 90381466 90381526 61 6 35 44 80 8097.30% 3 − 93754052 93754088 37 6 35 44 80 80 97.30% 3 − 9048640690486442 37 6 35 44 80 80 97.30% 2 − 94554961 94554997 37 6 35 12 72 8090.70% 19 − 27342003 27342234 232 6 35 44 80 80 100.00%16_KI270728v1_random − 1758745 1758952 208 6 35 44 80 80 97.30% 15 −19798346 19798382 37 6 35 44 80 80 97.30% 12 − 37264677 37264713 37 6 3344 80 80 94.60% 5_GL000208v1_random − 41930 41966 37 6 33 44 80 8094.60% 3 − 91530771 91530807 37 6 33 44 80 80 94.60% 3 − 9047415190474187 37 6 33 44 80 80 94.60% 3 − 90506100 90506136 37 6 33 44 80 8094.60% 22 − 15172797 15172833 37 6 33 44 80 80 94.60% 20 − 3016587030165906 37 6 33 44 80 80 94.60% 2 − 94553091 94553127 37 6 33 44 80 8094.60% 10 − 41830500 41830536 37 6 33 44 80 80 94.60% 10 − 4183558141835617 37 6 32 44 80 80 97.10% 11 − 48920295 48920502 208 6 32 44 7780 97.10% 10 − 41833723 41833756 34 6 31 44 80 80 91.90% X − 6252777662527812 37 6 31 44 80 80 91.90% 5 − 49627390 49627426 37 6 31 44 80 8091.90% 5 − 49627730 49627766 37 6 31 44 80 80 91.90% 5 − 4963273949632775 37 6 31 44 80 80 91.90% 5 − 49633079 49633115 37 6 31 44 80 8091.90% 3 − 90440780 90440816 37 6 31 44 80 80 91.90% 3 − 9047807590478111 37 6 31 44 80 80 91.90% 20 − 29721565 29721601 37 6 31 44 80 8091.90% 20 − 29721905 29721941 37 6 31 44 80 80 91.90% 15 − 1979495019794986 37 6 31 44 80 80 91.90% 14 − 18240976 18241012 37 6 31 44 80 8091.90% 11 − 48941375 48941411 37 6 31 44 80 80 91.90% 10 − 4183524141835277 37 6 29 44 80 80 89.20% 5_GL000208v1_random − 54691 54727 37 629 44 80 80 89.20% 20 − 29710536 29710572 37 6 29 44 80 80 89.20% 20 −30157815 30157851 37 6 29 46 80 80 91.50% 14 − 18244726 18244760 35 6 2944 80 80 89.20% 14 − 18261786 18261822 37 6 28 44 75 80 93.80% 3 −93717401 93717432 32 6 28 45 80 80 88.90% 3 − 90406941 90406976 36 6 2845 80 80 88.90% 22 − 15178081 15178116 36 6 28 47 80 80 91.20% 2 −94555812 94555845 34 6 28 44 75 80 93.80% 15 − 19799886 19799917 32 6 2644 75 80 90.70% 3 − 90398541 90398572 32 6 26 44 75 80 90.70% 20 −29705071 29705102 32 6 26 44 75 80 90.70% 20 − 28806396 28806427 32 6 2644 75 80 90.70% 11 − 48941721 48941752 32 6 25 44 72 80 93.20% 5 −46344317 46344345 29 6 23 44 72 80 89.70% 12 − 37644105 37644133 29 7 761 80 80 97.50% 3 − 93708268 93708347 80 7 76 1 80 80 97.50% 3 − 9371115393711232 80 7 76 1 80 80 97.50% 3 − 91545144 91545223 80 7 76 1 80 8097.50% 3 + 91663169 91663248 80 7 76 1 80 80 97.50% 3 + 9165771991657798 80 7 76 1 80 80 97.50% 3 + 91651933 91652012 80 7 76 1 80 8097.50% 3 + 91643254 91643333 80 7 76 1 80 80 97.50% 3 + 9163611291636191 80 7 76 1 80 80 97.50% 3 + 91627442 91627521 80 7 76 1 80 8097.50% 3 + 91621657 91621736 80 7 76 1 80 80 97.50% 3 + 9161587491615953 80 7 76 1 80 80 97.50% 3 + 91613319 91613398 80 7 76 1 80 8097.50% 3 + 91607539 91607618 80 7 76 1 80 80 97.50% 3 + 9160175491601833 80 7 76 1 80 80 97.50% 3 + 91598863 91598942 80 7 76 1 80 8097.50% 3 + 91595972 91596051 80 7 76 1 80 80 97.50% 3 + 9159274291592821 80 7 76 1 80 80 97.50% 3 + 91587808 91587887 80 7 76 1 80 8097.50% 3 + 91582023 91582102 80 7 74 1 80 80 96.30% 3 − 9154293491543013 80 7 74 1 80 80 96.30% 3 − 91547359 91547438 80 7 64 1 80 8090.00% 10 − 41838322 41838401 80 7 60 15 80 80 92.40% 3 − 9155125991551323 65 7 60 1 80 80 87.50% 3 − 90493548 90493627 80 7 57 1 69 8091.40% 5_GL000208v1_random − 15344 15412 69 7 57 1 69 80 91.40% 11 −55254272 55254340 69 7 56 1 70 80 91.20% 7 − 60887032 60888467 1436 7 561 72 80 87.20% 3 − 93733396 93733466 71 7 56 1 72 80 88.90% 19 −27410471 27410542 72 7 55 1 69 80 89.90% Un_KI270366v1 − 6689 6757 69 755 1 69 80 89.90% 5_GL000208v1_random − 51498 51566 69 7 55 1 69 8091.20% 3 − 93752224 93756891 4668 7 55 1 69 80 89.90% 3 − 9042814490428212 69 7 55 1 69 80 89.90% 15 − 19791369 19791437 69 7 54 1 68 8089.80% 3 − 93756309 93756376 68 7 54 1 72 80 87.50% 2 − 9453981494539885 72 7 53 1 69 80 88.50% 9 − 63096124 63096192 69 7 53 1 69 8088.50% 5_GL000208v1_random − 21197 21265 69 7 53 1 69 80 88.50%5_GL000208v1_random − 49619 49687 69 7 53 1 69 80 88.50% 5 − 4632904846329116 69 7 53 1 69 80 88.50% 5 − 46343839 46343907 69 7 53 1 69 8088.50% 5 − 47097837 47097905 69 7 53 1 69 80 88.50% 3 − 9051311490513182 69 7 53 1 69 80 88.50% 3 − 90441844 90441912 69 7 53 1 69 8088.50% 3 − 90418694 90418762 69 7 53 1 69 80 88.50% 22 − 1600751416007582 69 7 53 1 69 80 88.50% 15 − 19795163 19795231 69 7 53 1 69 8090.70% 11 − 48924424 48936523 12100 7 53 1 69 80 88.50% 1 − 124991106124991174 69 7 52 2 69 80 87.50% Un_KI270442v1 − 137105 137171 67 7 51 169 80 89.30% 5_GL000208v1_random − 13332 13727 396 7 51 1 69 80 87.00% 5− 46332969 46333037 69 7 51 1 69 80 87.00% 5 − 47091644 47091712 69 7 511 69 80 87.00% 3 − 90530039 90530107 69 7 51 1 69 80 87.00% 3 − 9040715490407222 69 7 51 1 69 80 87.00% 3 − 90290939 90291007 69 7 51 1 69 8088.10% 21 − 8048651 8048887 237 7 51 1 69 80 87.00% 20 − 2971751429717582 69 7 51 1 67 80 88.10% 2 − 94530091 94530157 67 7 51 1 69 8087.00% 11 − 50771954 50772022 69 7 51 1 69 80 87.00% 11 − 5075305950753127 69 7 51 1 70 80 87.20% 11 − 48922728 48924492 1765 7 51 1 69 8087.00% 11 − 48915933 48916001 69 7 51 1 69 80 87.00% 10 − 3943325639433324 69 7 50 1 72 80 84.80% 10 − 39454861 39454932 72 7 49 1 69 8085.60% Un_KI270442v1 − 369389 369457 69 7 49 7 69 80 85.50%5_GL000208v1_random − 20518 20579 62 7 49 7 69 80 85.50% 5 − 4709715847097219 62 7 49 1 69 80 85.60% 22 − 16083094 16083162 69 7 49 1 69 8085.60% 22 − 15199454 15199522 69 7 49 1 69 80 86.60% 20 − 2881493328815171 239 7 49 1 69 80 85.60% 15 − 19805889 19805957 69 7 49 1 69 8085.60% 12 − 37702094 37702162 69 7 49 1 69 80 87.50% 11 − 4891334448913412 69 7 49 1 69 80 85.60% 10 − 39425609 39425677 69 7 48 1 66 8086.40% 5 − 47088102 47088167 66 7 48 1 70 80 88.80% 21 − 7206506 7206917412 7 48 1 69 80 88.60% 20 − 29721094 29721333 240 7 48 1 69 80 82.90%12 − 37693536 37693602 67 7 47 1 70 80 94.40% 3 − 90437932 90445312 73817 47 1 69 80 84.10% 22 − 16082239 16082307 69 7 47 1 69 80 84.10% 22 −12333401 12333469 69 7 47 1 69 80 84.10% 19 − 27401084 27401152 69 7 471 69 80 84.10% 11 − 55247620 55247688 69 7 47 1 69 80 84.10% 11 −50752549 50752617 69 7 47 1 69 80 84.10% 11 − 50770589 50770657 69 7 471 69 80 84.10% 10 − 39426799 39426867 69 7 46 15 70 80 91.10% 3 −91524518 91524573 56 7 45 1 69 80 89.50% 3 − 90495431 90495667 237 7 451 69 80 82.70% 3 − 90389724 90389792 69 7 45 18 72 80 91.00% 16 −34615026 34615080 55 7 45 1 69 80 85.80% 12 − 37677473 37677712 240 7 451 69 80 82.70% 11 − 50727095 50727163 69 7 45 7 69 80 85.80% 1 −124995209 124995271 63 7 44 1 70 80 91.00% Un_KI270519v1 − 43678 4374770 7 42 1 69 80 77.80% 11 − 50719811 50719872 62 7 41 17 69 80 88.70% 3− 93735273 93735325 53 7 41 1 69 80 79.80% 11 − 50774189 50774257 69 738 1 69 80 86.60% 7 − 57908715 57908955 241 7 37 15 69 80 93.10% 2 −94531284 94531509 226 7 36 16 69 80 83.40% 22 − 16037827 16037880 54 729 1 42 80 89.20% 10 − 39347886 39347941 56 7 28 1 32 80 93.80% 11 −48934951 48934982 32 7 27 35 69 80 84.90% 21 − 12979571 12979604 34 7 2742 72 80 93.60% 11 − 55246591 55246621 31 7 26 1 32 80 90.70%5_GL000208v1_random − 76600 76631 32 7 24 42 69 80 92.90%5_GL000208v1_random − 76564 76591 28 7 22 42 69 80 89.30% 3 − 9375665093756677 28 7 20 6 29 80 91.70% 3 − 90577162 90577185 24 8 80 1 80 80100.00% 3 + 92383662 92383741 80 8 80 1 80 80 100.00% 3 + 9232154192321620 80 8 80 1 80 80 100.00% 3 + 92297210 92297289 80 8 80 1 80 80100.00% 3 + 92195963 92196042 80 8 80 1 80 80 100.00% 3 + 9214186392141942 80 8 80 1 80 80 100.00% 3 + 92127745 92127824 80 8 80 1 80 80100.00% 3 + 92124851 92124930 80 8 80 1 80 80 100.00% 3 + 9212196092122039 80 8 80 1 80 80 100.00% 3 + 92086923 92087002 80 8 80 1 80 80100.00% 3 + 92034340 92034419 80 8 80 1 80 80 100.00% 3 + 9197683991976918 80 8 80 1 80 80 100.00% 3 + 91879275 91879354 80 8 80 1 80 80100.00% 3 + 91831144 91831223 80 8 80 1 80 80 100.00% 3 + 9180834891808427 80 8 80 1 80 80 100.00% 3 + 91702033 91702112 80 8 80 1 80 80100.00% 3 + 91677709 91677788 80 8 76 1 80 80 97.50% 3 − 9370818893708267 80 8 76 1 80 80 97.50% 3 − 93711073 93711152 80 8 74 1 80 8096.30% 3 − 91545064 91545143 80 8 74 1 80 80 96.30% 3 − 9155117991551258 80 8 72 1 80 80 95.00% 3 − 91547279 91547358 80 8 62 1 80 8088.80% 3 − 91541335 91541414 80 8 62 1 80 80 88.80% 3 − 9154285491542933 80 8 58 1 80 80 86.30% 10 − 41838242 41838321 80 8 52 1 70 8087.20% 3 − 93714984 93715053 70 8 51 11 80 80 87.20% 2 − 9453392494553282 19359 8 49 1 61 80 90.20% 10 − 41840461 41840521 61 8 48 11 8080 84.30% 12 − 37369039 37369108 70 8 46 11 70 80 88.40% 12 − 3471257734712636 60 8 41 8 63 80 87.50% 3 − 90414354 90414410 57 8 41 8 63 8087.50% 12 − 37348170 37348226 57 8 39 11 69 80 91.70% 5 − 5019973950199971 233 8 39 11 61 80 88.30% 5 − 50240472 50240522 51 8 38 8 61 8085.20% 7 − 57988212 57988265 54 8 37 11 61 80 86.30% 7 − 6173434361734393 51 8 37 1 49 80 87.80% 3 − 93716528 93716576 49 8 37 11 61 8086.30% 3 − 91533117 91533167 51 8 37 18 68 80 86.30% 11 − 5504799955048049 51 8 35 11 61 80 84.40% X − 62523852 62523902 51 8 34 18 69 8082.70% 19 − 24215431 24215482 52 8 32 39 80 80 88.10% 18 − 2081834620818387 42 8 32 15 60 80 84.80% 12 − 37692273 37692318 46 8 32 8 49 8088.10% 11 − 48787030 48787071 42 8 32 11 61 80 90.00% 10 − 4182996141830351 391 8 31 11 63 80 79.30% 5_GL000208v1_random − 8823 8875 53 831 15 59 80 84.50% 5_GL000208v1_random − 3033 3077 45 8 31 36 74 8089.80% 20 − 28801250 28801288 39 8 31 11 49 80 89.80% 11 − 5520156655201604 39 8 30 39 74 80 91.70% 10 − 39453925 39453960 36 8 29 15 49 8091.50% 4 − 51798948 51798982 35 8 29 15 49 80 91.50% 4 − 5180778851807822 35 8 29 15 49 80 91.50% 4 − 51809998 51810032 35 8 29 11 49 8087.20% 4 − 51817125 51817163 39 8 29 11 49 80 94.00% 22 − 1123246411232502 39 8 29 36 69 80 94.20% 20 − 28804132 28804168 37 8 27 16 53 8070.00% 4 − 158252184 158252213 30 8 25 36 69 80 88.30% Un_KI270442v1 −151517 151553 37 8 25 36 68 80 87.90% 20 − 28798869 28798901 33 8 24 3663 80 92.90% 11 − 48798491 48798518 28 8 23 39 61 80 100.00% 16 −34614445 34614467 23 8 23 8 31 80 100.00% 12 − 37352449 37352473 25 8 228 29 80 100.00% 5_GL000208v1_random − 72600 72621 22 8 20 41 60 80100.00% 18 − 40667710 40667729 20 8 20 54 73 80 100.00% 13 − 8692179586921814 20 9 80 1 80 80 100.00% 3 + 91968245 91968324 80 9 80 1 80 80100.00% 3 + 91936439 91936518 80 9 80 1 80 80 100.00% 3 + 9192010491920183 80 9 80 1 80 80 100.00% 3 + 91914320 91914399 80 9 80 1 80 80100.00% 3 + 91907675 91907754 80 9 80 1 80 80 100.00% 3 + 9189058391890662 80 9 80 1 80 80 100.00% 3 + 91879355 91879434 80 9 80 1 80 80100.00% 3 + 91865590 91865669 80 9 80 1 80 80 100.00% 3 + 9181710491817183 80 9 80 1 80 80 100.00% 3 + 91802649 91802728 80 9 80 1 80 80100.00% 3 + 91787854 91787933 80 9 80 1 80 80 100.00% 3 + 9177050391770582 80 9 80 1 80 80 100.00% 3 + 91767608 91767687 80 9 80 1 80 80100.00% 3 + 91750259 91750338 80 9 80 1 80 80 100.00% 3 + 9162471091624789 80 9 80 1 80 80 100.00% 3 + 91585076 91585155 80 9 78 1 80 8098.80% 3 − 93708108 93708187 80 9 78 1 80 80 98.80% 3 − 9371099393711072 80 9 76 1 80 80 97.50% 3 − 91544984 91545063 80 9 76 1 80 8097.50% 3 − 91551099 91551178 80 9 72 1 80 80 95.00% 3 − 9154277491542853 80 9 69 1 77 80 94.90% 3 − 91547202 91547278 77 9 68 1 80 8092.50% 10 − 41838162 41838241 80 9 65 1 80 80 91.20% 3 − 9371641793716496 80 9 61 7 80 80 91.90% 20 − 30996304 30997229 926 9 60 1 80 8087.50% 3 − 93714894 93714973 80 9 58 7 77 80 92.60% 5 − 4633619146336430 240 9 58 1 77 80 84.00% 10 − 41833938 41834012 75 9 57 7 73 8094.10% 11 − 50798462 50798866 405 9 56 1 77 80 82.70% 10 − 4183579941835873 75 9 56 7 80 80 87.90% 1 − 124979335 124979408 74 9 54 7 80 8086.50% Un_KI270538v1 − 77535 77608 74 9 54 7 80 80 86.50% 14 − 1823571718235790 74 9 53 7 77 80 89.40% 20 − 30980980 30981219 240 9 53 7 80 8086.50% 11 − 55248815 55249571 757 9 52 17 80 80 90.70% X − 6250937262509435 64 9 52 7 69 80 92.10% 5 − 46329059 46333382 4324 9 52 7 77 8082.70% 2 − 94532312 94532380 69 9 51 7 77 80 87.90% 20 − 2971242229712659 238 9 51 1 69 80 87.00% 12 − 37268438 37268506 69 9 51 7 80 8085.20% 1 − 124958310 124985720 27411 9 51 8 80 80 85.00% 1 − 124984453124984525 73 9 50 7 77 80 81.20% 2 − 94536067 94536135 69 9 50 7 77 8081.20% 12 − 34709777 34709845 69 9 50 7 77 80 90.40% 11 − 4892374648925359 1614 9 50 7 77 80 81.20% 10 − 41839860 41839928 69 9 50 7 80 8083.80% 1 − 124953535 124953608 74 9 49 17 77 80 92.90% 11 − 4894210648942335 230 9 49 7 80 80 93.00% 1 − 124980015 124988447 8433 9 48 7 7780 79.80% 2 − 94552109 94552177 69 9 48 7 68 80 88.80% 16 − 3420954434209605 62 9 48 7 66 80 90.00% 12 − 37637165 37637224 60 9 48 17 77 8084.80% 12 − 34714537 34714595 59 9 48 17 80 80 87.50% 1 − 124986840124986903 64 9 47 7 76 80 79.50% 20 − 29721271 29721338 68 9 47 7 69 8087.40% 12 − 37636478 37636540 63 9 46 17 77 80 83.10% 22 − 1517130815171366 59 9 46 17 77 80 92.50% 20 − 30981322 30981550 229 9 46 7 77 8091.00% 14 − 18259788 18260030 243 9 46 7 68 80 87.10% 12 − 3763988937639950 62 9 46 17 77 80 83.10% 11 − 48931341 48931399 59 9 46 17 77 8083.10% 10 − 39439540 39439598 59 9 43 7 69 80 84.20% 20 − 2972093429720996 63 9 43 7 71 80 83.10% 2 − 94538971 94539035 65 9 42 24 80 8093.90% 1 − 124996404 124996801 398 9 41 7 69 80 82.60% 11 − 4893903848939100 63 9 40 17 66 80 90.00% 16_KI270728v1_random − 1758459 175850850 9 40 7 61 80 91.70% 12 − 37268617 37268840 224 9 40 17 68 80 88.50%10 − 39425279 39425330 52 9 39 17 59 80 95.40% 22 − 16039594 16039636 439 39 17 73 80 84.30% 21 − 7971575 7971631 57 9 39 17 73 80 84.30% 21 −7975490 7975546 57 9 39 17 73 80 84.30% 21 − 7979400 7979456 57 9 39 1773 80 84.30% 21 − 7995565 7995621 57 9 39 17 73 80 84.30% 21 − 80321558032211 57 9 39 7 69 80 89.80% 2 − 94521902 94522135 234 9 39 7 61 8085.50% 2 − 94537276 94537330 55 9 39 7 61 80 85.50% 12 − 3794706637947120 55 9 39 17 67 80 88.30% 11 − 48911480 48911530 51 9 39 19 69 8088.30% 1 − 124969980 124970030 51 9 38 7 48 80 95.30% 5 − 4632942146329462 42 9 38 6 69 80 91.20% 12 − 34712328 34712556 229 9 38 8 69 8089.60% 1 − 124938226 124938459 234 9 38 8 69 80 89.60% 1 − 124939756124939989 234 9 37 17 69 80 85.00% 20 − 29707997 29708049 53 9 37 21 6980 87.80% 12 − 34834973 34835021 49 9 36 22 61 80 95.00% 4 − 5179580151795840 40 9 36 22 61 80 95.00% 4 − 51798011 51798050 40 9 36 8 61 8083.40% 1 − 124936876 124936929 54 9 34 22 61 80 92.50% 4 − 5180685151806890 40 9 34 22 61 80 92.50% 4 − 51809061 51809100 40 9 34 21 77 8074.60% 14 − 18261832 18261886 55 9 33 17 80 80 88.60% 1 − 124987694124987755 62 9 26 7 36 80 93.40% 20 − 29701231 29701260 30 9 26 7 36 8093.40% 2 − 94534230 94534259 30 9 26 7 36 80 93.40% 17 − 2688283426882863 30 9 26 7 36 80 93.40% 15 − 19794865 19794894 30 9 26 7 36 8093.40% 14 − 18231331 18231360 30 9 26 7 36 80 93.40% 14 − 1823627518236304 30 9 26 7 36 80 93.40% 1 − 124934857 124934886 30 9 25 7 35 8093.20% 3 − 90455999 90456027 29 9 25 8 36 80 93.20% 12 − 3735785237357880 29 9 24 7 36 80 90.00% 2 − 94555388 94555417 30 9 21 23 43 80100.00% 16 − 38265826 38265846 21 9 21 23 43 80 100.00% 16 − 3827600538276025 21 9 20 17 36 80 100.00% 20 − 30995835 30995854 20 9 20 17 3680 100.00% 2 − 94513376 94513395 20 9 20 17 36 80 100.00% 11 − 4893121048931229 20 9 20 17 36 80 100.00% 1 − 124979888 124979907 20

Although a number of sequences of each oligo had 100% homology tochromosome 3, there are also many off-target hits. For example, as shownin Table 2, Oligo 1 had 16 on-target hits, but also 24 off-target hits;Oligo 3 had 16 on-target hits, but also 33 off-target hits, etc.

TABLE 2 # on-target hits (CHR3) # off-target hits (on other SEQ ID 100%unless otherwise chromosomes) NO specified >85% identity (>70 nt) 1 1624 2 16 20 3 16 33 4 16 8 5 16 34 6 15 5 7 18 (no 100%, >97%) 3 8 16 2 916 3 10 18 1 11 15 20 12 19 15 13 16 23 14 18 (no 100%, >95%) 2 15 18 016 17 (no 100%, >98%) 6 17 16 33 18 16 (no 100%, >98%) 1

These results strongly suggest that the centromere region of chromosome3 may not contain specific sequences because there are many off-targethits to other chromosomes. Notwithstanding this, the present inventorshave surprisingly identified 18 oligonucleotide sequences that hybridizehighly specifically to the centromere region of chromosome 3, such thatblocking DNA can be eliminated in hybridization assays if so desired.

I. DEFINITIONS

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.”

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which the disclosure pertains are described in various generaland more specific references, including, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates, 1992 (andSupplements to 2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Conjugating, joining, bonding or linking: Covalently linking onemolecule to another molecule to make a larger molecule. For example,making two polypeptides into one contiguous polypeptide molecule, orcovalently attaching a mass tag, hapten, nucleic acid, or other moleculeto a polypeptide, such as a scFv antibody.

Detectable label: A compound or composition that is conjugated directlyor indirectly to another molecule (such as a nucleic acid probe) tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent and fluorogenic moieties, chromogenicmoieties, haptens, affinity tags, and radioactive isotopes. The labelcan be directly detectable (e.g., optically detectable) or indirectlydetectable (for example, via interaction with one or more additionalmolecules that are in turn detectable). Exemplary labels in the contextof the probes disclosed herein are described below. Methods for labelingnucleic acids, and guidance in the choice of labels useful for variouspurposes, are discussed, e.g., in Sambrook and Russell, in MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress (2001) and Ausubel et al., in Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Intersciences (1987, andincluding updates).

Hapten: A molecule, typically a small molecule that can combinespecifically with an antibody, but typically is substantially incapableof being immunogenic except in combination with a carrier molecule

Hybridization: To form base pairs between complementary regions of twostrands of DNA, RNA, or between DNA and RNA, thereby forming a duplexmolecule. Hybridization conditions resulting in particular degrees ofstringency will vary depending upon the nature of the hybridizationmethod and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (such as the Na+ concentration) of the hybridization bufferwill determine the stringency of hybridization. The presence of achemical which decreases hybridization (such as formamide) in thehybridization buffer will also determine the stringency (Sadhu et al.,J. Biosci. 6:817-821, 1984). Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning, second edition, ColdSpring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11).Hybridization conditions for ISH are also discussed in Landegent et al.,Hum. Genet. 77:366-370, 1987; Lichter et al., Hum. Genet. 80:224-234,1988; and Pinkel et al., Proc. Natl. Acad. Sci. USA 85:9138-9142, 1988.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, or cell) has been substantially separated or purifiedaway from other biological components in a preparation, a cell of anorganism, or the organism itself, in which the component occurs, such asother chromosomal and extra-chromosomal DNA and RNA, proteins and cells.Nucleic acid molecules and proteins that have been “isolated” includenucleic acid molecules and proteins purified by standard purificationmethods. The term also embraces nucleic acid molecules and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acid molecules and proteins. In some examples, thenucleic acid probes disclosed herein are isolated nucleic acid probes.

Linker: As used herein, a linker is a molecule or group of atomspositioned between two moieties. For example, a mass tag conjugate mayinclude a linker between the mass tag and the specific binding moiety.Typically, linkers are bifunctional, i.e., the linker includes afunctional group at each end, wherein the functional groups are used tocouple the linker to the two moieties. The two functional groups may bethe same, i.e., a homobifunctional linker, or different, i.e., aheterobifunctional linker.

Multiplex, -ed, -ing: Embodiments of the present invention allowmultiple targets in a sample to be detected substantiallysimultaneously, or sequentially, as desired, using plural differentconjugates. Multiplexing can include identifying and/or quantifyingnucleic acids generally, DNA, RNA, peptides, proteins, both individuallyand in any and all combinations. Multiplexing also can include detectingtwo or more of a gene, a messenger and a protein in a cell in itsanatomic context.

Phosphatidylinositol 3-kinase, p110 subunit (PIK3CA): Also known asphosphoinositide-3-kinase, catalytic, alpha polypeptide. Humanphosphatidylinositol 3-kinase (EC 2.7.1.137) is composed of 85-kD and110-kD subunits. The 85-kD subunit lacks phosphatidylinositol 3-kinaseactivity and acts as an adaptor, coupling the 110-kD subunit (p110) toactivated protein tyrosine kinases. The human p110 subunit is referredto herein as PIK3CA. Hiles et al. (Cell 70:419-429, 1992) found that thehuman cDNA for p110 predicts a 1,068-amino acid protein related to aprotein which in S. cerevisiae is involved in the sorting of proteins tothe vacuole. In COS-1 cells, p110 was catalytically active only whencomplexed with p85-alpha. Volinia et al. (Genomics 24:472-477, 1994)contributed to the structural and functional understanding ofphosphatidylinositol 3-kinase by purifying, cloning, and subsequentlyelucidating the expression of the bovine enzyme. cDNA for the humanPIK3CA encodes a protein 99% identical to the bovine p110. Thechromosomal localization of the gene for human PIK3CA is shown to be at3q21-qter as determined using somatic cell hybrids. In situhybridization performed using Alu-PCR from the YAC DNA located the humangene in 3q26.3 [Chromosome 3: 178,865,902-178,957,881]. The sequence forthe human PIK3CA gene was disclosed as early as 1994 by Volinia et al.

Probe: A nucleic acid molecule that is capable of hybridizing with atarget nucleic acid molecule (e.g., genomic target nucleic acidmolecule) and, when hybridized to the target, is capable of beingdetected either directly or indirectly. Thus probes permit thedetection, and in some examples quantification, of a target nucleic acidmolecule. In particular examples, a probe includes at least two segmentscomplementary to uniquely specific nucleic acid sequences of a targetnucleic acid molecule and are thus capable of specifically hybridizingto at least a portion of the target nucleic acid molecule. Generally,once at least one segment or portion of a segment has (and remains)hybridized to the target nucleic acid molecule other portions of theprobe may (but need not) be physically constrained from hybridizing tothose other portions' cognate binding sites in the target (e.g., suchother portions are too far distant from their cognate binding sites);however, other nucleic acid molecules present in the probe can bind toone another, thus amplifying signal from the probe. A probe can bereferred to as a “labeled nucleic acid probe,” indicating that the probeis coupled directly or indirectly to a detectable moiety or “label,”which renders the probe detectable.

Sample: A specimen containing DNA (for example, genomic DNA), RNA(including mRNA), protein, or combinations thereof, obtained from asubject. Examples include, but are not limited to, chromosomalpreparations, peripheral blood, urine, saliva, tissue biopsy, fineneedle aspirate, surgical specimen, bone marrow, amniocentesis samples,and autopsy material. In one example, a sample includes genomic DNA. Insome examples, the sample is a cytogenetic preparation, for examplewhich can be placed on microscope slides. In particular examples,samples are used directly, or can be manipulated prior to use, forexample, by fixing (e.g., using formalin).

Sequence identity: The identity (or similarity) between two or morenucleic acid sequences is expressed in terms of the identity orsimilarity between the sequences. Sequence identity can be measured interms of percentage identity; the higher the percentage, the moreidentical the sequences are. Sequence similarity can be measured interms of percentage similarity (which takes into account conservativeamino acid substitutions); the higher the percentage, the more similarthe sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8:155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology and on the Internet, foruse in connection with the sequence analysis programs blastp, blastn,blastx, tblastn and tblastx. Additional information can be found at theNCBI web site. BLASTN may be used to compare nucleic acid sequences,while BLASTP may be used to compare amino acid sequences. If the twocompared sequences share homology, then the designated output file willpresent those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences. The BLAST-like alignment tool(BLAT) may also be used to compare nucleic acid sequences (Kent, GenomeRes. 12:656-664, 2002). BLAT is available from several sources,including Kent Informatics (Santa Cruz, Calif.) and on the Internet(genome.ucsc.edu).

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1554 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 15 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(that is, 15÷20*100=75).

Subject: Any multi-cellular vertebrate organism, such as human ornon-human mammals (e.g., veterinary subjects).

Target nucleic acid sequence or molecule: A defined region or particularportion of a nucleic acid molecule, for example a portion of a genome(such as a gene or a region of mammalian genomic DNA containing a geneof interest). In an example where the target nucleic acid sequence is atarget genomic sequence, such a target can be defined by its position ona chromosome (e.g., in a normal cell), for example, according tocytogenetic nomenclature by reference to a particular location on achromosome; by reference to its location on a genetic map; by referenceto a hypothetical or assembled contig; by its specific sequence orfunction; by its gene or protein name; or by any other means thatuniquely identifies it from among other genetic sequences of a genome.In some examples, the target nucleic acid sequence is mammalian genomicsequence (for example human genomic sequence).

In some examples, alterations of a target nucleic acid sequence (e.g.,genomic nucleic acid sequence) are “associated with” a disease orcondition. In some examples, detection of the target nucleic acidsequence can be used to infer the status of a sample with respect to thedisease or condition. For example, the target nucleic acid sequence canexist in two (or more) distinguishable forms, such that a first formcorrelates with absence of a disease or condition and a second (ordifferent) form correlates with the presence of the disease orcondition. The two different forms can be qualitatively distinguishable,such as by polynucleotide polymorphisms, and/or the two different formscan be quantitatively distinguishable, such as by the number of copiesof the target nucleic acid sequence that are present in a cell.

Uniquely specific sequence: A nucleic acid sequence (for example, asequence of at least of at least 20 basepairs (bp) (such as at least 20bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, or more)that is present only one time in a haploid genome of an organism. In aparticular example, a uniquely specific nucleic acid sequence is anucleic acid sequence from a target nucleic acid that has 100% sequenceidentity with the target nucleic acid and has no significant identity toany other nucleic acid sequences present in the specific haploid genomethat includes the target nucleic acid.

Vector: Any nucleic acid that acts as a carrier for other (“foreign”)nucleic acid sequences that are not native to the vector. Whenintroduced into an appropriate host cell a vector may replicate itself(and, thereby, the foreign nucleic acid sequence) or express at least aportion of the foreign nucleic acid sequence. In one context, a vectoris a linear or circular nucleic acid into which a nucleic acid sequenceof interest is introduced (for example, cloned) for the purpose ofreplication (e.g., production) and/or manipulation using standardrecombinant nucleic acid techniques (e.g., restriction digestion). Avector can include nucleic acid sequences that permit it to replicate ina host cell, such as an origin of replication. A vector can also includeone or more selectable marker genes and other genetic elements known inthe art. Common vectors include, for example, plasmids, cosmids, phage,phagemids, artificial chromosomes (e.g., BAC, PAC, HAC, YAC), andhybrids that incorporate features of more than one of these types ofvectors. Typically, a vector includes one or more unique restrictionsites (and in some cases a multi-cloning site) to facilitate insertionof a target nucleic acid sequence.

II. SYSTEMS FOR IN SITU HYBRIDIZATION FOR CHROMOSOME ENUMERATION A.Chromosome 3

The most common target for a control region of chromosome 3 (CHR3) ISHis the centromeric region. The centromeric regions of all humanchromosomes are characterized by distinct subsets of a diverse tandemlyrepeated DNA family, alpha satellite. Since alpha satellite DNA clustersmost often contain monomer variants that differ from the consensussequence by up to 40%, blocking DNA is usually included with the probesto suppress sequences contained within the target loci that are commonto other chromosomes.

Single-stranded probes directed to the control region (centromericregion) of chromosome 3 were designed that achieved acceptable signalintensity levels and background levels within 1 hour of hybridization(see FIG. 4) and without the use of blocking DNA. For example, theprobes are configured to achieve a staining intensity of greater than orequal to 2 and staining coverage of greater than or equal to 50% ofnuclei. Also designed were single-stranded probes directed to a targetregion near and within the PIK3CA gene locus that also achievedacceptable signal intensity levels and background levels within 1 hourof hybridization (see FIG. 4) and without the use of blocking DNA. Thecriteria in Table 3 were used to evaluate whether the ISH assay isacceptable or not acceptable.

TABLE 3 Acceptable (A) Not Acceptable (N) Signal 3, Signals are brightand easily 1, Specific signals are visible but too Intensity identifiedin >80% of cells within the weak to reliably identify in ≧50% of targetregion. the targeted region. 2, Specific signals are sufficiently 0.5,Signals are visible but absent or intense to reliably identify in >50%too weak to reliably identify in 80% of of cells within the targetedregion. cells. 0, Signals are not visible. Background 1, Backgroundsignals (either punctate 3, Background signals (punctate signals ordiffuse, hazy staining) are signals, diffuse staining, haze) coverpresent but are sufficiently weak in 75-100% of cells within the targetintensity within the nuclei to permit region and are sufficientlyintense to reliable identification of specific obscure specific signals.signals in >50% of cells within the 2, Background signals (punctatetarget region. signals, diffuse staining, haze) cover 0, Backgroundstaining is not 50-75% of cells within the target observed in >80% ofcells within the region and are sufficiently intense to target region.obscure specific signals

From the perspective of manufacturing and quality control, asingle-stranded probe having an exact structure are more reproduciblymanufactured using oligonucleotide synthesis compared to the approachesbased on PCR, nick translation, or other random synthetic approaches.From the perspective of cost analysis, the probes that do not requireblocking DNA provide for a less expensive assay.

The present disclosure describes systems for ISH featuring a controlprobe specific to a control region of a chromosome, e.g., a centromeretarget of a chromosome. The chromosome detected may be chromosome 3, orany other appropriate chromosome. The control probe is configured toachieve a staining intensity of greater than or equal to 2 and stainingcoverage of greater than or equal to 50% of the number of nuclei within3 hours when applied to a control sample (e.g., as described above,TABLE 3). In some embodiments, the present invention achieves a stainingcoverage of ≧55% of the number of nuclei within 3 hours, e.g., ≧60% ofthe number of nuclei, ≧65% of the number of nuclei, ≧70% of the numberof nuclei, ≧75% of the number of nuclei, ≧80% of the number of nuclei,≧85% of the number of nuclei, ≧90% of the number of nuclei.

In some embodiments, the systems for ISH also feature a target probespecific for a target region (e.g., for detecting a target gene) on thecorresponding chromosome.

In some embodiments, the control probe comprises a first plurality(e.g., a plurality of a single probe, a plurality of different probessuch as a set or pool of probes) of single-stranded oligonucleotideprobes. One or more of the plurality of probes may comprise a sequenceselected from the group consisting of SEQ ID NOs: 1-18 (see Table 4below). In some embodiments, one or more of the first plurality ofprobes comprise a truncated version (e.g., at least 30 contiguous bp, atleast 35 contiguous bp, at least 40 contiguous bp, at least 45contiguous bp, at least 50 contiguous bp, at least 55 contiguous bp, atleast 60 contiguous bp, at least 65 contiguous bp, at least 70contiguous bp, at least 75 contiguous bp, etc.) of one of the sequencesin Table 4 (SEQ ID NOs: 1-18). In some embodiments, one or more of thefirst plurality of probes comprises a sequence that has at least 70%sequence identity, at least 75% sequence identity, at least 80% sequenceidentity, at least 85% sequence identity, at least 90% sequenceidentity, or at least 95% sequence identity to one of the sequences inTable 4 (SEQ ID NOs: 1-18). The first plurality of single-strandedoligonucleotide probes is configured to hybridize uniquely andspecifically to a portion of the control region of human chromosome 3 sothat other chromosomes or portions thereof are not evidently labeled.

As used herein, reference to use of SEQ ID NOs: 1-18 may also includethe use of complementary sequences of SEQ ID NOs: 1-18.

In some embodiments, the probes target between 2 and 18 distinctportions within the control region. In some embodiments, the probestarget between 4 and 18 distinct portions within the control region. Insome embodiments, the probes target between 6 and 18 distinct portionswithin the control region. In some embodiments, the probes targetbetween 8 and 18 distinct portions within the control region. In someembodiments, the probes target between 10 and 18 distinct portionswithin the control region. In some embodiments, the probes targetbetween 12 and 18 distinct portions within the control region. In someembodiments, the probes target between 14 and 18 distinct portionswithin the control region. In some embodiments, the probes targetbetween 16 and 18 distinct portions within the control region. In someembodiments, the probes target between 2 and 12 distinct portions withinthe control region. In some embodiments, the probes target between 4 and12 distinct portions within the control region. In some embodiments, theprobes target between 6 and 12 distinct portions within the controlregion. In some embodiments, the probes target between 8 and 12 distinctportions within the control region. In some embodiments, the probestarget between 10 and 12 distinct portions within the control region.

TABLE 4 SEQ ID NO: Sequences Length 1TCTGTGGAATTTGCAAGGGGAGATTTCAAGCACTTTGAGGC 80CATTGGTGGAAAAGGAAATATCTTCGTATGAAAACTAGA 2CAGAATCATTCTCAGGAACTACTTTGTGATATGTGCATTCA 80ACTCCCAGAGTTTAACCTTTCTTTTCATAGATGAGTTTG 3AATGCTAGACAGAAGAATTCTCAGTAACTTCTTTTGGGATG 80TATGTATTCAAATCAGAGAGTTGAACCTTCCTTTAGACA 4GAGCGGATTGGAAACACTCTTTTTGTGGAATTTGCAAGTGG 80AAAATTCTAGCAGTATGAGGCCAATGGTACAAAAGGAAA 5TATCTTCGTATAAAAACTAGACAGTATCATTCTCAGAAACT 80GCTTTGTGATGTGTGTATTAAACTCACAGAGTTGAACAT 6TTCTTTGCATAGAGCAGTTTGGAAAGACTTAGTTTGTGCAG 80TGTGCAAGTGGATATTTGGAACTCTTTGAGGCCTTCGTT 7ATGTGTGCATTCGGCTCACAGAGTTGAACCTTACTTTGGAA 80AGAGCAGTTTTCTAACACTCTTTTTGTAAAAGTTCCAAG 8TGAATACTTTGAGTGCTTTGAAGCCTACGGTTGACAACGAA 80ATATCTTCCTGTAAAAACTACAAAGAATCATTCGCAGAA 9ACCACGTTGTGATCTCTGCATTCAACTCACAGAGTTCAACC 80TTTCTTCCTATAGAGCAGTTATGAAACAGTCTCTTTGTA 10GAATTTGCAAGGGTGTATTTAGAGGGCATTGAAGCCTACG 80GTAGAAAAGGAAATATCTTACCATAAAATCTAGTCAGAAG 11CATTCTCAGCAACTGAGTTGTGATGTTTGCATTCAACTCAC 80AGAGTTCAACATTCCTTTTAATGGAGCGGTTTTGAAACA 12ATTGGAAACACTCTTTTTGTGGAATTTTCAGGTGGAGGTAT 80CAAGCGCTTTGAGGCCAATGATAGAAAAGGAAATACCTT 13CGTATAATAATTAGACGGAATCATTCTCAGAAACCGCTTTG 80CAATGTGTGCGTTCAACTCACAGTGTTTAACCTTTCTTT 14TCATACAAGTTGTTTCGAAACACTCTTTTTGCAGAATCTGC 80AAGTGGATATTTGGACCTCTTTGAAGTCTTCGTTGGAAA 15GCCTTCGTAGTAAACGGGATTTCTTCGTGTAATGATAGACA 80ATAGAATTCTCAGTGAATTTTTTTCTGTGTGTGTGTATT 16TTGAGTGAATGGTAGGAAAGGAAATACCTTCGTATAAAA 80ACTAGACGGAGTCATTCTCAGAAACTACTTTGTGATGTTT 17ATTTGCAAGTGGAGAATTCTAGCGCTTTGACGCCAATGGTA 80GAAAGGAAATATCTTCGTATAAAAACTAGACAGTATCAT 18CGGTTGGAAACACTTTTTGTGGAATTTTCAGGGGGAGACTT 80CAAGCGCTTTGAAGTGAATGGTAGGAAAGGAAATACCTT

The first plurality of single-stranded oligonucleotide probes may beconstructed in a variety of lengths. For example, in some embodiments,the probes each comprise between 40 to 100 nucleotides. In someembodiments, the probes each comprise between 50 to 100 nucleotides. Insome embodiments, the probes each comprise between 60 to 110nucleotides. In some embodiments, the probes each comprise between 40 to120 nucleotides. In some embodiments, the probes each comprise at least40 nucleotides. In some embodiments, the probes each comprise at least50 nucleotides. In some embodiments, the probes each comprise at least60 nucleotides. In some embodiments, the probes each comprise at least70 nucleotides.

The present invention also features slides with a plurality of nucleistained for a chromosome control, e.g., CHR3 control. The slide may becontacted with one or more of the above systems (e.g., probes). Theslide features enumerable signals indicative of the number of chromosome3 centromere regions present in a cell, e.g., cells should exhibit twocopies of the CHR3 centromere normally.

In some embodiments, more than 50% of the nuclei have enumerable signalsfor the chromosome. An enumerable signal may be a generally round shape.The round shape can be defined as shown in FIGS. 14A and 14B, wherein around shape is a simple closed curve that fits within a first region,the first region lies on and outside an inner circle and on and inside aconcentric outer circle, the inner circle has an inner radius (R_(in))and the outer circle has an outer radius (R_(out)), wherein the simpleclose curve has a radius R_(simple), wherein R_(in)≦R_(simple)≦R_(out),and wherein, R_(in) is ≧50% of R_(out). One aspect of the round shape isthat it is a condensed signal compared to a diffuse signal. Thehybridization of the probe to the target may not form a round signal ata very high magnification, but with a chromogenic deposition and atlower magnifications (e.g. those customary within the anatomic pathologylaboratory, 100×, 600×) the signal appears round. The parameters of agenerally round shape are specified so as to distinguish the signalsassociated with the present probes to those previously possible. Anotheraspect of the present disclosure is that the extent to which the signalsare in a generally round shape, condensed, and regular, the ability toread the signals by either a pathologist or using a machine reader, isenhanced.

In some embodiments, the inner radius is no less than 40% of the outerradius. In some embodiments, the inner radius is no less than 50% of theouter radius. In some embodiments, the inner radius is no less than 55%of the outer radius. In some embodiments, the inner radius is no lessthan 60% of the outer radius. In some embodiments, the inner radius isno less than 65% of the outer radius. In some embodiments, the innerradius is no less than 70% of the outer radius. In some embodiments, theinner radius is no less than 75% of the outer radius. In someembodiments, the inner radius is no less than 80% of the outer radius.In some embodiments, the inner radius is no less than 85% of the outerradius. In some embodiments, the inner radius is no less than 90% of theouter radius.

In some embodiments, more than 60% of the nuclei have enumerable signalsfor the chromosome. In some embodiments, more than 70% of the nucleihave enumerable signals for the chromosome. In some embodiments, morethan 80% of the nuclei have enumerable signals for the chromosome. Insome embodiments, more than 90% of the nuclei have enumerable signalsfor the chromosome. The nuclei may not be enumerable if the tissuesectioning process has destroyed that portion of the cell, if thatportion of the cell is divided between two slides, or if that portion ofthe cell is wholly within a separate slide. The nuclei may also beenumerable if the tissue condition prevents probe penetration to thespecific binding site (i.e. the cell is not sufficiently accessible tothe probe) or if the target region of DNA is substantially degraded.

In some embodiments, the sum of the surface area covered by stainingsignal is calculated and assigned a 100% value, and at least 50% of thesum of the surface area is derived from discrete round signals (or roundshapes).

A round shape can be defined as shown in FIGS. 14A and 14B, wherein around shape is a simple closed curve that fits within a first region,the first region lies on and outside an inner circle and on and inside aconcentric outer circle, the inner circle has an inner radius (R_(in))and the outer circle has a outer radius (R_(out)), wherein the simpleclose curve has a radius R_(simple), wherein R_(in)≦R_(simple)≦R_(out),and wherein, R_(in) is ≧50% of R_(out).

In some embodiments, the inner radius is no less than 50% of the outerradius. In some embodiments, more than 60% of said sum of the surfacearea is derived from discrete round signals. In some embodiments, morethan 70% of said sum of the surface area is derived from discrete roundsignals. In some embodiments, the inner radius is no less than 60% ofthe outer radius. In some embodiments, the inner radius is no less than75% of the outer radius. In some embodiments, the inner radius is noless than 90% of the outer radius.

Referring to FIG. 12-13, the radii (e.g., outer radii) of a plurality ofsignals were measured. In some embodiments, the outer radius is betweenabout 0.25 to 0.675 μm. In some embodiments, the outer radius is betweenabout 0.2 to 0.75 μm. In some embodiments, the outer radius is betweenabout 0.15 to 1 μm. In some embodiments, the average outer radius of theenumerable signals is between about 0.2 to 0.75 μm. In some embodiments,the average outer radius of the enumerable signals has a standarddeviation of less than 0.5 μm. In some embodiments, the average outerradius of the enumerable signals has a standard deviation of less than0.25 μm.

In some embodiments, the enumerable round signals are mono-sized. Asused herein, a population of “mono-sized” round signals have theR_(simple) being within 15% plus or minus of each other. In someembodiments, the population of “mono-sized” round signals have theR_(simple) being within 10% plus or minus of each other. In someembodiments, the population of “mono-sized” round signals have theR_(simple) being within 5% plus or minus of each other.

B. Target Gene (PIK3CA)

In some embodiments, the systems for ISH also feature a target probespecific for a target region (e.g., for detecting a target gene, forgene copy enumeration) on the corresponding chromosome.

The target region may comprise the PIK3CA gene locus (or nearbynucleotides). Disclosed herein are probes directed to the human PIK3CAgene (e.g., Gene ID No. 5290; NC_000003.11 (178866311 . . . 178952500)),which is incorporated herein by reference as present in GENBANK® on Apr.30, 2012) and depicted in FIG. 1. As described below in detail inEXAMPLE 1, the PIK3CA target probe is specific to a region betweennucleotides 178,640,071 and 179,399,807 of human chromosome 3.

In some embodiments, the target probe comprises a second plurality(e.g., a plurality of a single probe, a plurality of different probessuch as a set or pool of probes) of single-stranded oligonucleotideprobes. One or more of the plurality of probes may comprise a sequenceselected from the group consisting of SEQ ID NOs: 19-1230 (see FIGS. 14Aand 14B). The second plurality of single-stranded oligonucleotide probesis configured to hybridize uniquely and specifically to a portion of thetarget region of the corresponding chromosome so that other genes orchromosomes or portions thereof are not evidently labeled.

III. KITS

Also disclosed are kits including one or more of the oligonucleotideprobes (for example, one or more of SEQ ID NOs: 1-18). For example, kitscan include at least one probe (such as at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more probes) or at least one probe set (such as at least 1, 2,3, 4, or 5 probe sets) as described herein. In one example, the kitcomprises probes such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17 or all of SEQ ID NOs: 1-18 (or sequences at least60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least90% identical to SEQ ID NOs: 1-18; or truncated versions of SEQ ID NOs:1-18). In other examples, the probes (or the probe set) are in a singlecontainer.

The kits may also comprise one or more reagents for detecting the probe(for example, by in situ hybridization), or for producing a detectablylabeled probe. For example, a kit can include at least one of thedisclosed nucleic acid probes or probe sets, along with one or morebuffers, labeled dNTPs, a labeling enzyme (such as a polymerase),primers, nuclease free water, and instructions for producing a labeledprobe. In another example, the kit includes one or more of the disclosednucleic acid probes (unlabeled or labeled) along with buffers and otherreagents for performing in situ hybridization. For example, if one ormore unlabeled probes are included in the kit, labeling reagents canalso be included, along with specific detection agents (for example,fluorescent, chromogenic, luminescent and/or radiometric) and otherreagents for performing an in situ hybridization assay, such as paraffinpretreatment buffer, protease(s) and protease buffer, prehybridizationbuffer, hybridization buffer, wash buffer, counterstain(s), mountingmedium, or combinations thereof. In some examples, such kit componentsare present in separate containers. The kit can optionally furtherinclude control slides (such as positive or negative controls) forassessing hybridization and signal of the probe(s).

In certain examples, the kits include avidin, antibodies, and/orreceptors (or other anti-ligands). Optionally, one or more of thedetection agents (including a primary detection agent, and optionally,secondary, tertiary or additional detection reagents) are labeled, forexample, with a hapten or fluorophore (such as a fluorescent dye orquantum dot). In some instances, the detection reagents are labeled withdifferent detectable moieties (for example, different fluorescent dyes,spectrally distinguishable quantum dots, different haptens, etc.). Forexample, a kit can include two or more nucleic acid probes or probe setsthat correspond to and are capable of hybridizing to different targetnucleic acids (for example, any of the target nucleic acids disclosedherein). The first probe or probe set can be labeled with a firstdetectable label (e.g., hapten, fluorophore, etc.), the second probe orprobe set can be labeled with a second detectable label, and anyadditional probes or probe sets (e.g., third, fourth, fifth, etc.) canbe labeled with additional detectable labels. The first, second, and anysubsequent probes or probe sets can be labeled with different detectablelabels, although other detection schemes are possible. If the probe(s)are labeled with indirectly detectable labels, such as haptens, the kitscan include detection agents (such as labeled avidin, antibodies orother specific binding agents) for some or all of the probes. In oneembodiment, the kit includes probes and detection reagents suitable formultiplex ISH.

In one example, the kit also includes an antibody conjugate, such as anantibody conjugated to a label (e.g., an enzyme, fluorophore, orfluorescent nanoparticle). In some examples, the antibody is conjugatedto the label through a linker, such as PEG, 6×-His, streptavidin, orGST.

IV. DETECTABLE LABELS AND METHODS OF LABELING

The probes disclosed herein may comprise one or more labels (e.g., atleast 1 at least 2, at least 3, at least 4, at least 5, at least 6,etc.), for example to permit detection of the probe/nucleic acidsequence (or region) of interest. In various applications, such as insitu hybridization procedures, a nucleic acid probe includes a label(e.g., a detectable label). A “detectable label” is a molecule ormaterial that can be used to produce a detectable signal that indicatesthe presence or concentration of the probe (particularly the bound orhybridized probe) in a sample. Thus, a labeled nucleic acid moleculeprovides an indicator of the presence or quantity (for example, genecopy number) of a target nucleic acid (to which the labeled uniquelyspecific nucleic acid molecule is bound or hybridized) in a sample. Thedisclosure is not limited to the use of particular labels, althoughexamples are provided.

A label associated with one or more nucleic acid molecules (such as thedisclosed probes) can be detected either directly or indirectly. A labelcan be detected by any known or yet to be discovered mechanism includingabsorption, emission and/or scattering of a photon (including radiofrequency, microwave frequency, infrared frequency, visible frequencyand ultra-violet frequency photons). Detectable labels include colored,fluorescent, phosphorescent and luminescent molecules and materials,catalysts (such as enzymes) that convert one substance into anothersubstance to provide a detectable difference (such as by converting acolorless substance into a colored substance or vice versa, or byproducing a precipitate or increasing sample turbidity), haptens thatcan be detected by antibody binding interactions, and paramagnetic andmagnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules(or fluorochromes). Numerous fluorochromes are known to those of skillin the art, and can be selected, for example from Life Technologies,e.g., see, The Handbook—A Guide to Fluorescent Probes and LabelingTechnologies. Examples of particular fluorophores that can be attached(for example, chemically conjugated) to a nucleic acid molecule (such asa uniquely specific binding region) are provided in U.S. Pat. No.5,866,366 to Nazarenko et al., such as4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamine-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2′,7′-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone;ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B,sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA);tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);riboflavin; rosolic acid and terbium chelate derivatives.

Other suitable fluorophores include thiol-reactive europium chelates,which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem.248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP,Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described inU.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Otherfluorophores known to those skilled in the art can also be used, forexample those available from Life Technologies (Carlsbad, Calif.) andincluding the ALEXA FLUOR® series of dyes (for example, as described inU.S. Pat. Nos. 5,696,157, 6,130,101 and 6, 716,979), the BODIPY seriesof dyes (dipyrrometheneboron difluoride dyes, for example as describedin U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854,5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative ofthe sulfonated pyrene described in U.S. Pat. No. 5,132,432) and MarinaBlue (U.S. Pat. No. 5,830,912). In addition to the fluorochromesdescribed above, a fluorescent label can be a fluorescent nanoparticle,such as a semiconductor nanocrystal, e.g., a quantum dot. Additionallabels include, for example, radioisotopes (such as 3H), metal chelatessuch as DOTA and DPTA chelates of radioactive or paramagnetic metal ionslike Gd3+, and liposomes.

Detectable labels that can be used with nucleic acid molecules (such asthe disclosed probes) also include enzymes, for example horseradishperoxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucoseoxidase, β-galactosidase, β-glucuronidase, or β-lactamase. Where thedetectable label includes an enzyme, a chromogen, fluorogenic compound,or luminogenic compound can be used in combination with the enzyme togenerate a detectable signal (numerous of such compounds arecommercially available, for example, from Life Technologies). Particularexamples of chromogenic compounds include diaminobenzidine (DAB),4-nitrophenylphosphate (pNPP), fast red, fast blue, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue,and tetrazolium violet.

Alternatively, an enzyme can be used in a metallographic detectionscheme. For example, silver in situ hybridization (SISH) proceduresinvolve metallographic detection schemes for identification andlocalization of a hybridized genomic target nucleic acid sequence.Metallographic detection methods include using an enzyme, such asalkaline phosphatase, in combination with a water-soluble metal ion anda redox-inactive substrate of the enzyme. The substrate is converted toa redox-active agent by the enzyme, and the redox-active agent reducesthe metal ion, causing it to form a detectable precipitate. (See, forexample, U.S. Pat. No. 7,632,652,). Metallographic detection methodsalso include using an oxido-reductase enzyme (such as horseradishperoxidase) along with a water soluble metal ion, an oxidizing agent anda reducing agent, again to form a detectable precipitate. (See, forexample, U.S. Pat. No. 6,670,113).

In non-limiting examples, the disclosed nucleic acid probes are labeledwith dNTPs covalently attached to hapten molecules (such as anitro-aromatic compound (e.g., 2,4-dinitrophenyl (DNP)), biotin,fluorescein, digoxigenin (DIG), etc.). Additional haptens suitable forlabeling the disclosed probes include nitropyrazole,3-hydroxyquinoxaline, thiazolesulfonamide, nitrocinnamic acid, rotenone,7-(diethylamino)coumarin-3-carboxylic acid, benzodiazepine, orbenzofuran haptens (see, e.g., International Pat. Publ. No. WO2012/003476 incorporated herein by reference). Methods for conjugatinghaptens and other labels to dNTPs (e.g., to facilitate incorporationinto labeled probes) are well known in the art. For examples ofprocedures, see, e.g., U.S. Pat. Nos. 5,258,507, 4,772,691, 5,328,824,and 4,711,955. Indeed, numerous labeled dNTPs are availablecommercially, for example from Life Technologies (Carlsbad, Calif.). Alabel can be directly or indirectly attached to a dNTP at any locationon the dNTP, such as a phosphate (e.g., α, β or γ phosphate) or a sugar.

Detection of labeled nucleic acid molecules can be accomplished bycontacting the hapten-labeled nucleic acid molecules bound to thegenomic target nucleic acid with a primary anti-hapten antibody. In oneexample, the primary anti-hapten antibody (such as a mouse anti-haptenantibody) is directly labeled with an enzyme. In another example, asecondary anti-species antibody (such as a goat anti-mouse IgG antibody)conjugated to an enzyme is used for signal amplification. In chromogenicin situ hybridization CISH a chromogenic substrate is added, for SISH,silver ions and other reagents as outlined in the referencedpatents/applications are added.

In some examples, a probe is labeled by incorporating one or morelabeled dNTPs using an enzymatic (polymerization) reaction. For example,the disclosed nucleic acid probes (for example, incorporated into aplasmid vector) can be labeled by nick translation (using, for example,biotin, DNP, digoxigenin, etc.) or by random primer extension withterminal transferase (e.g., 3′ end tailing). In some examples, thenucleic probe is labeled by a modified nick translation reaction wherethe ratio of DNA polymerase I to deoxyribonuclease I (DNase I) ismodified to produce greater than 100% of the starting material. Inparticular examples, the nick translation reaction includes DNApolymerase I to DNase I at a ratio of at least about 800:1, such as atleast 2000:1, at least 4000:1, at least 8000:1, at least 10,000:1, atleast 12,000:1, at least 16,000:1, such as about 800:1 to 24,000:1 andthe reaction is carried out overnight (for example, for about 16-22hours) at a substantially isothermal temperature, for example, at about16° C. to 25° C. (such as room temperature). If the probe is included ina probe set (for example, multiple plasmids, such as 2, 3, 4, 5, 6, 7,8, 9, 10, or more plasmids), the plasmids may be mixed in an equal molarratio prior to performing the labeling reaction (such as nicktranslation or modified nick translation).

In other examples, chemical labeling procedures can also be employed.Numerous reagents (including hapten, fluorophore, and other labelednucleotides) and other kits are commercially available for enzymaticlabeling of nucleic acids, including the disclosed nucleic acid probes.As will be apparent to those of skill in the art, any of the labels anddetection procedures disclosed above are applicable in the context oflabeling a probe, e.g., for use in in situ hybridization reactions. Forexample, the Amersham MULTIPRIME® DNA labeling system, various specificreagents and kits available from Molecular Probes/Life Technologies, orany other similar reagents or kits can be used to label the nucleicacids disclosed herein. In particular examples, the disclosed probes canbe directly or indirectly labeled with a hapten, a ligand, a fluorescentmoiety (e.g., a fluorophore or a semiconductor nanocrystal), achromogenic moiety, or a radioisotope. For example, for indirectlabeling, the label can be attached to nucleic acid molecules via alinker (e.g., PEG or biotin). Additional methods that can be used tolabel probe nucleic acid molecules are provided in U.S. Pat. No.7,541,455.

V. METHODS FOR IN SITU HYBRIDIZATION FOR CHROMOSOME ENUMERATION

The present invention also features in situ hybridization (ISH) assays,e.g., bright-field ISH assays, for detection of a gene target and achromosome (e.g., centromere target of a chromosome) using single-strandoligonucleotide probes. For example, a method comprises contacting atissue sample with a control probe specific to a control region of achromosome (e.g., chromosome 3), wherein the control probe is asingle-stranded oligonucleotide probe labeled with at least one firstlabel. The control probe may be configured to achieve a stainingintensity of ≧2 and staining coverage of ≧50% of nuclei within 3 hourswhen applied to a control sample. The method further compriseshybridizing the control probe to the control region under conditions fora period of time less than about 3 hours (e.g., ≦about 2.5 hours, ≦about2 hours, ≦about 1.5 hour, or ≦about 1 hour), rinsing the sample toremove unbound probe, and detecting the presence of the hybridizedprobe.

In some embodiments, the method further comprises contacting the tissuesample with a target probe specific to a target region (e.g., PIK3CA) ofthe chromosome, wherein the target probe is a single-strandedoligonucleotide probe labeled with at least one second label.

In some embodiments, the method further comprises applying chromogenicdetection reagents that recognize the first label and amplify the signalassociated with said first label. The method may feature the use of oneor more probes (e.g., SEQ ID NOs: 1-18) or systems as described herein.

Genome-specific blocking DNA (such as human DNA, for example, totalhuman placental DNA or Cot-1™ DNA) is usually included in ahybridization solution (such as for in situ hybridization) to suppressprobe hybridization to repetitive DNA sequences or to counteract probehybridization to highly homologous (frequently identical) off targetsequences when a probe complementary to a human genomic target nucleicacid is utilized. In hybridization with standard probes, in the absenceof genome-specific blocking DNA, an unacceptably high level ofbackground staining (for example, non-specific binding, such ashybridization to non-target nucleic acid sequence) is usually present,even when a “repeat-free” probe is used. The disclosed nucleic acidprobes exhibit reduced background staining, even in the absence ofblocking DNA. In particular examples, the hybridization solutionincluding the disclosed probes does not include genome-specific blockingDNA (for example, total human placental DNA or Cot-1™ DNA, if the probeis complementary to a human genomic target nucleic acid). This advantageis derived from the uniquely specific nature of the target sequencesincluded in the nucleic acid probe; each labeled probe sequence bindsonly to the cognate uniquely specific genomic sequence. This results indramatic increases in signal to noise ratios for ISH techniques.

As such, some methods herein may be free from the use of blocking DNA.However, in some embodiments, blocking DNA may be used. In someembodiments, an amount of blocking DNA is used but the amount ofblocking DNA is sufficient to block out no more than a specified percentof the non-specific binding, e.g., no more than 50%, 40%, 30%, 20%, or10%.

In order to determine an amount of blocking DNA that is sufficient toblock out no more than a specified percent (e.g., 50%) of thenon-specific binding, the following tests may be conducted. Set up an insitu hybridization assay, contact a tissue sample with a double strand(e.g. DNA) control probe specific to a control region of a chromosome(in combination with zero to a serially, gradually increasing amount ofblocking DNA); hybridize the double strand control probe to the controlregion; rinse the sample to remove unbound double strand probe; anddetect the presence of the hybridized probe. Then observe the amount ofbackground that is blocked by the serially increasing blocking DNA ineach assay. The amount of blocking DNA that achieves a specified percentof the blocking of the background corresponds to the amount of blockingDNA that is sufficient to block out no more than a specified percent(e.g., 50%) of the non-specific binding. For example, the amount ofblocking DNA that achieves blocking out 50% of percent of the backgroundcorresponds to the amount of blocking DNA that is sufficient to blockout no more than 50% of the non-specific binding.

In some embodiments, said amount of blocking DNA is between about 1pg/ml to 1 mg/ml. In some embodiments, said amount of blocking DNA isbetween about 1 pg/ml to 0.5 mg/ml. In some embodiments, said amount ofblocking DNA is between about 1 pg/ml to 0.25 mg/ml. In someembodiments, said amount of blocking DNA is between about 1 pg/ml to 1μg/ml.

In some illustrative embodiments, methods for obtaining two bright-fieldchromogenic in situ hybridization signals per cell may comprisecontacting a tissue sample containing a plurality of cells with acontrol probe specific to a control region of a single chromosome, theprobe selected so as to not evidently bind non-specifically in theabsence of blocking DNA; hybridizing the control probe to the controlregion of said chromosome; rinsing the sample to remove unbound probe;and detecting the presence of the hybridized probe via a chromogenicreagent so as to generate two bright-field chromogenic in situhybridization signals per cell. In order to determine that the selectedprobe does not evidently bind non-specifically in the absence ofblocking DNA, a comparative assay (Assay 2) may be conducted along sidewith the aforementioned assay (Assay 1), wherein the same selected probeis employed in both Assay 1 and Assay 2. Assay 1 is free of the blockingDNA and Assay 2 employs a blocking DNA. Then the respective data of thetwo assays are compared. The selected probe does not evidently bindnon-specifically in the absence of blocking DNA when the data of the tworespective assays are the same or substantially the same.

In some examples the hybridization solution may contain carrier DNA froma different organism (for example, salmon sperm DNA or herring spermDNA, if the genomic target nucleic acid is a human genomic targetnucleic acid) to reduce non-specific binding of the probe to non-DNAmaterials (for example to reaction vessels or slides) with high netpositive charge which can non-specifically bind to the negativelycharged probe DNA.

Methods of the present invention may comprise detecting signals whereinmore than 50% of the nuclei of the tissue sample have enumerable signalsfor said chromosome, wherein an enumerable signal is a generally roundshape (e.g., as described above). In some embodiments, backgroundsignals are not observed in >70% of cells of the tissue sample. In someembodiments, background signals are not observed in >80% of cells of thetissue sample. In some embodiments, background signals are not observedin >90% of cells of the tissue sample. In some embodiments, backgroundsignals are present but are sufficiently weak in intensity so as topermit identification of enumerable signals in >50% of the nuclei.

In some embodiments, more than 60% of the nuclei have enumerablechromosome signals. In some embodiments, more than 70% of the nucleihave enumerable chromosome signals. In some embodiments, the innerradius is no less than 60% of the outer radius. In some embodiments, theinner radius is no less than 75% of the outer radius. In someembodiments, the inner radius is no less than 90% of the outer radius.

In situ hybridization (ISH) involves contacting a sample containing atarget nucleic acid (e.g., a genomic target nucleic acid) in the contextof a metaphase or interphase chromosome preparation (such as a cell ortissue sample mounted on a slide) with a labeled probe specificallyhybridizable or specific for the target nucleic acid (for example, oneor more of the probes disclosed herein). The slides are optionallypretreated, e.g., to remove paraffin or other materials that caninterfere with uniform hybridization. The chromosome sample and theprobe are both treated, for example by heating to denature the doublestranded nucleic acids. The probe (formulated in a suitablehybridization buffer) and the sample are combined, under conditions andfor sufficient time to permit hybridization to occur (typically to reachequilibrium). The chromosome preparation is washed to remove excessprobe, and detection of specific labeling of the target is performedusing standard techniques.

For example, a biotinylated probe can be detected usingfluorescein-labeled avidin or avidin-alkaline phosphatase. Forfluorochrome detection, the fluorochrome can be detected directly, orthe samples can be incubated, for example, with fluoresceinisothiocyanate (FITC)-conjugated avidin. Amplification of the FITCsignal can be effected, if necessary, by incubation withbiotin-conjugated goat anti-avidin antibodies, washing and a secondincubation with FITC-conjugated avidin. For detection by enzymeactivity, samples can be incubated, for example, with streptavidin,washed, incubated with biotin-conjugated alkaline phosphatase, washedagain and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).The enzyme reaction can be performed in, for example, AP buffercontaining NBT/BCIP and stopped by incubation in 2×SSC. For a generaldescription of in situ hybridization procedures, see, e.g., U.S. Pat.No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. Forexample, procedures for performing FISH are described in U.S. Pat. Nos.5,447,841; 5,472,842; and 5,427,932 CISH is described in U.S. Pat. No.6,942,970, and additional detection methods are provided in U.S. Pat.No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunctionwith FISH, CISH, and SISH procedures to improve sensitivity, resolution,or other desirable properties. As discussed above, probes labeled withfluorophores (including fluorescent dyes and quantum dots) can bedirectly optically detected when performing FISH. Alternatively, theprobe can be labeled with a non-fluorescent molecule, such as a hapten(such as the following non-limiting examples: biotin, digoxigenin, DNP,and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans,triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-basedcompounds, Podophyllotoxin, Podophyllotoxin-based compounds, andcombinations thereof), ligand or other indirectly detectable moiety.Probes labeled with such non-fluorescent molecules (and the targetnucleic acid sequences to which they bind) can then be detected bycontacting the sample (e.g., the cell or tissue sample to which theprobe is bound) with a labeled detection reagent, such as an antibody(or receptor, or other specific binding partner) specific for the chosenhapten or ligand. The detection reagent can be labeled with afluorophore (e.g., quantum dot) or with another indirectly detectablemoiety, or can be contacted with one or more additional specific bindingagents (e.g., secondary or specific antibodies), which can in turn belabeled with a fluorophore. Optionally, the detectable label is attacheddirectly to the antibody, receptor (or other specific binding agent).

Alternatively, the detectable label is attached to the binding agent viaa linker, such as a hydrazide thiol linker, a polyethylene glycollinker, or any other flexible attachment moiety with comparablereactivities. For example, a specific binding agent, such as anantibody, a receptor (or other anti-ligand), avidin, or the like can becovalently modified with a fluorophore (or other label) via aheterobifunctional polyalkyleneglycol linker such as aheterobifunctional polyethyleneglycol (PEG) linker. A heterobifunctionallinker combines two different reactive groups selected, e.g., from acarbonyl-reactive group, an amine-reactive group, a thiol-reactive groupand a photo-reactive group, the first of which attaches to the label andthe second of which attaches to the specific binding agent.

In other examples, the probe, or specific binding agent (such as anantibody, e.g., a primary antibody, receptor or other binding agent) islabeled with an enzyme that is capable of converting a fluorogenic orchromogenic composition into a detectable fluorescent, colored orotherwise detectable signal (e.g., as in deposition of detectable metalparticles in SISH). As indicated above, the enzyme can be attacheddirectly or indirectly via a linker to the relevant probe or detectionreagent. Examples of suitable reagents (e.g., binding reagents) andchemistries (e.g., linker and attachment chemistries) are described inU.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and2007/0117153.

In further examples, a signal amplification method is utilized, forexample, to increase sensitivity of the probe. For example, tyramidesignal amplification may be utilized (See U.S. Pat. No. 5,196,306). Inone variation of this method a biotinylated nucleic acid probe detectsthe presence of a target by binding thereto. Next astreptavidin-peroxidase conjugate is added. The streptavidin binds tothe biotin. A substrate of biotinylated tyramide (tyramine is4-(2-aminoethyl)phenol) is used, which presumably becomes a free radicalwhen interacting with the peroxidase enzyme. The phenolic radical thenreacts quickly with the surrounding material, thus depositing or fixingbiotin in the vicinity. This process is repeated by providing moresubstrate (biotinylated tyramide) and building up more localized biotin.Finally, the “amplified” biotin deposit is detected with streptavidinattached to a fluorescent molecule. Alternatively, the amplified biotindeposit can be detected with avidin-peroxidase complex, that is then fed3,3′-diaminobenzidine to produce a brown color. It has been found thattyramide attached to fluorescent molecules also serve as substrates forthe enzyme, thus simplifying the procedure by eliminating steps. Yetanother amplification approach is described in U.S. Patent Publ. No.2013/0260379.

In other examples, the signal amplification method utilizes branched DNA(bDNA) signal amplification. In some examples, target-specificoligonucleotides (label extenders and capture extenders) are hybridizedwith high stringency to the target nucleic acid. Capture extenders aredesigned to hybridize to the target and to capture probes, which areattached to a microwell plate. Label extenders are designed to hybridizeto contiguous regions on the target and to provide sequences forhybridization of a preamplifier oligonucleotide. Signal amplificationthen begins with preamplifier probes hybridizing to label extenders. Thepreamplifier forms a stable hybrid only if it hybridizes to two adjacentlabel extenders. Other regions on the preamplifier are designed tohybridize to multiple bDNA amplifier molecules that create a branchedstructure. Finally, alkaline phosphatase (AP)-labeled oligonucleotides,which are complementary to bDNA amplifier sequences, bind to the bDNAmolecule by hybridization. The bDNA signal is the chemiluminescentproduct of the AP reaction See, e.g., Tsongalis, Microbiol. Inf. Dis.126:448-453, 2006; U.S. Pat. No. 7,033,758.

In further examples, the signal amplification method utilizespolymerized antibodies. In some examples, the labeled probe is detectedby using a primary antibody to the label (such as an anti-DIG oranti-DNP antibody). The primary antibody is detected by a polymerizedsecondary antibody (such as a polymerized HRP-conjugated secondaryantibody or an AP-conjugated secondary antibody). The enzymatic reactionof AP or HRP leads to the formation of strong signals that can bevisualized.

It will be appreciated by those of skill in the art that byappropriately selecting labeled probe-specific binding agent pairs,multiplex detection schemes can be produced to facilitate detection ofmultiple target nucleic acids (e.g., genomic target nucleic acids) in asingle assay (e.g., on a single cell or tissue sample or on more thanone cell or tissue sample). For example, a first probe that correspondsto a first target nucleic acid can be labeled with a first hapten, suchas biotin, while a second probe that corresponds to a second targetnucleic acid can be labeled with a second hapten, such as DNP. Followingexposure of the sample to the probes, the bound probes can be detectedby contacting the sample with a first specific binding agent (in thiscase avidin labeled with a first fluorophore, for example, a firstspectrally distinct quantum dot, e.g., that emits at 585 nm) and asecond specific binding agent (in this case an anti-DNP antibody, orantibody fragment, labeled with a second fluorophore (for example, asecond spectrally distinct quantum dot, e.g., that emits at 705 nm).Additional probes/binding agent pairs can be added to the multiplexdetection scheme using other spectrally distinct fluorophores. Numerousvariations of direct, and indirect (one step, two step or more) can beenvisioned, all of which are suitable in the context of the disclosedprobes and assays.

Additional details regarding certain detection methods, e.g., asutilized in CISH and SISH procedures, can be found in Bourne, TheHandbook of Immunoperoxidase Staining Methods, published by DakoCorporation, Santa Barbara, Calif.

Difficulties frequently encountered in ISH testing may result from themanner in which the tissues are typically preserved. The mainstay of thediagnostic pathology laboratory has been for many decades theformalin-fixed, paraffin-embedded block of tissue, sectioned and mountedupon glass slides. Fixation in such a preservative causes cross-linkingof macromolecules, both amino acids and nucleic acids. Thesecross-linked components must be removed to allow access of the probe tothe target nucleic acid and to allow the antibody to recognize thecorresponding antigen. “Unmasking” the antigen and/or nucleic acid istypically accomplished manually with multiple pretreatment, proteolyticdigestion, and wash steps. Prior to or staining, complete removal of theparaffin is also required so that it does not interfere with antibody orprobe binding. Deparaffinization may be achieved by the use of multiple(e.g., two or three) successive clearing reagents that are paraffinsolvents (e.g., xylene, xylene substitutes, or toluene).

In some embodiments, preparing the sample includes the step of cellconditioning. Cell conditioning is discussed in greater detail in U.S.Pat. No. 6,855,552, Towne, et al. “Automated immunohistochemical and insitu hybridization assay formulations”, the subject matter of which isexpressly incorporated by reference. In illustrative cell conditioningsteps, a cell conditioning reagent is applied and the sample iscontacted at the appropriate temperature for an appropriate duration oftime so that the antigens and/or nucleic acid targets are sufficientlyexpressed for detection. One aspect of the present disclosure is thatthe automated instrument can automatically adjust the cell conditioningduration and/or temperature in response to the user inputs. Cellconditioning may further include applying a protease reagent.Illustratively, a protease treatment may involve the step of contactinga protease solution to a biological sample. The protease treatment, aswith cell conditioning, is intended to increase the expression of targetantigens and/or nucleic acids.

Cell conditioning reagents such as ethylenediaminetetraacetic acid(EDTA) for nucleic acid targets (ISH) may be used. The contacting may bedone at a temperature of about 95° C. for between about 2 and about 90minutes. A partial list of possible reagents appears in AnalyticalMorphology, Gu, ed., Eaton Publishing Co. (1997) at pp. 1-40. Sodiumdodecyl sulfate (SDS) and/or ethylene glycol may be included in theconditioning solution. Furthermore, metal ions or other materials may beadded to these reagents to increase effectiveness of the cellconditioning. Exemplary cell conditioning solutions are available fromVentana Medical Systems, Inc., Tucson, Ariz. (Cell Conditioning 1 (CC1)catalog #: 950-124; Cell Conditioning 2 (CC2) catalog #: 950-123; SSC(10×) catalog #: 950-110; ULTRA Cell Conditioning (ULTRA CC1) catalog #:950-224; ULTRA Cell Conditioning (ULTRA CC2) catalog #: 950-223,Protease 1 catalog #: 760-2018; Protease 2 catalog #: 760-2019; Protease3 catalog #: 760-2020). In some embodiments, applying the in situhybridization binding reagent occurs subsequent to applying the cellconditioning reagent and prior to applying the chromogenic reagent.

In illustrative embodiments, the method includes applying a rinsingreagent. Between various steps described herein and as part of thesystem described herein, rinse steps may be added to remove unreactedresidual reagents from the prior step. Rinse steps may further includeincubations, which include maintaining a rinsing reagent on the samplefor a pre-determined time at a pre-determined temperature with orwithout mixing. The conditions appropriate for the rinsing steps may bedistinct between the various steps. Exemplary rinsing reagents areavailable from Ventana Medical Systems, Inc., Tucson, Ariz. (ReactionBuffer (10×) catalog #: 950-300; Special Stains Wash (10×) catalog #:860-015).

Exemplary automated systems available through Ventana Medical Systems,Inc., Tucson, Ariz. include SYMPHONY® Staining System, catalog #:900-SYM3, VENTANA® BenchMark Automated Slide Preparation Systems,catalog #s: N750-BMKXT-FS, N750-BMKU-FS, VENTANA, and VENTANA® BenchMarkSpecial Stains automated slide stainer. These systems employ amicroprocessor controlled system including a revolving carouselsupporting radially positioned slides. A stepper motor rotates thecarousel placing each slide under one of a series of reagent dispenserspositioned above the slides. Bar codes on the slides and reagentdispensers permits the computer controlled positioning of the dispensersand slides so that different reagent treatments can be performed foreach of the various tissue samples by appropriate programming of thecomputer.

While EXAMPLE 1 below describes a single-stranded oligonucleotide-basedPIK3CA/CHR3 dual ISH assay, it is understood that those of ordinaryskill in the art could apply the discoveries disclosed herein to othergene/centromere combination of interest.

In some embodiments, the disclosed systems (e.g., probes) can be used inmethods of determining the copy number of a target nucleic acid (such asPIK3CA) in a biological sample (such as a tissue sample). Methods ofdetermining the copy number of a gene or chromosomal region are wellknown to those of skill in the art. In some examples, the methodsinclude in situ hybridization (such as fluorescent, chromogenic, orsilver in situ hybridization), comparative genomic hybridization, orpolymerase chain reaction (such as real-time quantitative PCR). In someexamples, methods of determining gene copy number include counting thenumber of ISH signals (such as fluorescent, colored, or silver spots)for the target nucleic acid in one or more individual cells. The methodsmay also include counting the number of ISH signals (such asfluorescent, colored, or silver spots) for a reference (such as achromosome-specific probe) in the cells. In particular examples, thenumber of copies of the gene (or chromosome) may be estimated by theperson (or computer, in the case of an automated method) scoring theslide. In some examples, an increased copy number relative to a control(such as an increase of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold,20-fold, or more relative to a control sample or reference value)indicates an increase in the target nucleic acid copy number.

In some examples, the method includes counting the number of copies percell or nucleus of a reference, such as a chromosomal locus known not tobe abnormal, for example a centromere. In some examples, the referenceis on the same chromosome as the gene of interest. Exemplary referencechromosomes that can be used for particular human genes of interest areprovided in Table 5. In particular examples, the reference locus isdetected by using a centromere-specific probe. Such probes are known inthe art and are commercially available, for example, Vysis CEP probes(Abbott Molecular, Des Plaines, Ill.) and SPOTLIGHT centromeric probes(Invitrogen, Carlsbad, Calif.). In some examples, a ratio of targetnucleic acid copy number to reference copy number greater than about two(such as greater than about 2, 3, 4, 5, 10, 20, or more) indicates anincrease in the target nucleic acid copy number.

TABLE 5 EXEMPLARY REFERENCE CHROMOSOMES FOR PARTICULAR TARGET NUCLEICACIDS Target Nucleic Acid Reference Chromosome PTEN 10 PIK3CA 3 TOP2A 17MET 7 MDM2 12

VI. METHODS OF SCORING

The present invention also features methods of scoring gene copy numberof a target region and optionally comparing it to the copy number of acontrol region. For additional methods of scoring, which may be usedwith the methods described herein, reference is made to U.S. Publ. Appl.No. 2012/0141472, which is hereby incorporated by reference fordisclosure related to scoring ISH.

In some examples, an increased gene copy number includes the gene copynumber per nucleus (such as average gene copy number per nucleus) in thesample of greater than about two copies of the gene per nucleus (such asgreater than 2, 3, 4, 5, 10, or 20 copies). In other examples, anincreased gene copy number includes a ratio of gene copy number to itscorresponding chromosome copy number (such as an average gene:chromosomeratio) in the sample of greater than about 2 (such as a ratio of greaterthan 2, 3, 4, 5, 10, or 20). In further examples, an increased gene copynumber includes an increase in gene copy number relative to a control(such as an increase of about 1.5-fold, about 2-fold, about 3-fold,about 5-fold, about 10-fold, about 20-fold, or more). Therefore, in someexamples, the method includes comparing the gene copy number in thesample from the subject to the gene copy number in a control or areference value or range of values expected for the gene copy number inan appropriate normal tissue.

Also disclosed herein is a method of scoring (for example, enumerating)copy number of a gene in a sample from a subject, wherein the sample isstained by ISH (such as FISH, SISH, CISH, or a combination of two ormore thereof) for the gene of interest and wherein individual copies ofthe gene are distinguishable in cells in the sample. In particularexamples, the sample is a biological sample from a subject, such as atumor sample (for example, a tumor biopsy). Methods of determining genecopy number by ISH are well known in the art.

In some embodiments, the method includes identifying individual cells ina sample with the highest number of signals per nucleus for the gene(such as the strongest signal in the sample), counting the number ofsignals for the gene in the identified cells, and determining an averagenumber of signals per cell, thereby scoring the gene copy number in thesample. In additional embodiments, the method further includes countingthe number of signals for a reference (such as a chromosomal locus knownnot to be abnormal, for example, centromeric DNA) and determining anaverage ratio of the number of signals for the gene to the number ofsignals for the reference per cell.

The scoring method may include identifying individual cells in thesample (such as a tissue section or tumor core) having the highestnumber of signals (such as the highest number of spots per cell or thebrightest intensity of staining) for the gene of interest in the cellsin the sample. Thus, the disclosed method may not determine gene copynumber in a random sampling of cells in the sample. Rather, the methodmay include specifically counting gene copy number in those cells thathave the highest gene copy number in the sample. In some examples,identifying the individual cells having the highest number of signalsfor the gene includes examining a sample stained by ISH for the geneunder low power microscopy (such as about 20><magnification). Cells withthe strongest signal (for example, highest amplification signal underhigher power) are identified for counting by eye or by an automatedimaging system. In some examples, such as when the sample is a tissuesection, the sample is examined (for example, visually scanned) toidentify a region that has a concentration of tumor cells that hasamplification of the gene. Gene copy number in the cells with highestamplification in the selected region is then counted. In other examples,such as when the sample is a tumor core (such as a tumor microarray),most of the sample is visible in the field of view under low powermagnification and the individual cells (such as tumor cells) with thestrongest signal (for example, highest amplification signal under highpower) are separately identified for counting. In particular examples,the cells chosen for counting the gene copy number may benon-consecutive cells, such as cells that are not adjacent to or incontact with one another. In other examples, at least some of the cellschosen for counting the gene copy number may be consecutive cells, suchas cells that are adjacent to or in contact with one another.

The disclosed methods may include counting the number of ISH signals(such as fluorescent, colored, or silver spots) for the gene in theidentified cells. The methods may also include counting the number ofISH signals (such as fluorescent, colored or silver spots) for areference (such as a chromosome-specific probe) in the identified cells.In some examples, the number of spots per cells is distinguishable inthe identified cells and the number of spots are counted (or enumerated)and recorded. In other examples, one or more of the identified cells mayinclude a cluster, which is the presence of multiple overlapping signalsin a nucleus that cannot be counted (or enumerated). In particularexamples, the number of copies of the gene (or chromosome) may beestimated by the person (or computer, in the case of an automatedmethod) scoring the slide. For example, one of skill in the art ofpathology may estimate that a cluster contains a particular number ofcopies of a gene (such as 10, 20, or more copies) based on experience inenumerating gene copy number in a sample. In other examples, thepresence of a cluster may be noted as a cluster, without estimating thenumber of copies present in the cluster.

The number of cells identified for counting is a sufficient number ofcells that provides for detecting a change (such as an increase ordecrease) in gene copy number. In some examples, the number of cellsidentified for counting is at least about 20, for example, at least 25,30, 40, 50, 75, 100, 200, 500, 1000 cells, or more. In a particularexample, about 50 cells are counted. In other examples, every cell inthe sample or every cell in a microscope field of vision, or in a numberof microscope fields (such as at least 2 microscope fields, at least 3,at least 4, at least 5, at least 6 microscope fields, and the like)which contains 3 or more copies of the gene of interest (such as 3, 4,5, 6, 7, 8, 9, 10, 15, 20, or more) is counted.

Methods may feature obtaining a sample having undergone ISH according tomethods disclosed herein. An area of neoplastic nuclei with the mostcopy numbers is identified and the enumerable signals for thechromosome/target are counted in 50-100 neoplastic nuclei and either 50adjacent mesenchymal nuclei or 50 adjacent normal epithelial nuclei.

Scoring criteria may be as follows: no staining or <1 dot/10 cells isscored as 0; 1-3 dots/cell is scored as 1; 4-9 dots/cell, none or veryfew dot clusters is scored as 2; 10-15 dots/cell and <10% dots are inclusters is scored as 3; and >15 dots/cell and >10% dots are in clustersis scored as 4.

In some embodiments, the average number of target signals (e.g., PIK3CA)per nuclei is calculated. In some embodiments, the average number ofchromosome (e.g., CHR3) copies per nuclei is calculated. In someembodiments, the target signal to chromosome signal ratio is calculated.

The disclosure is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1—Automated Bright-Field PIK3CA Copy Number Enumerationwith Oligo Dual In Situ Hybridization and its Correlation withOverexpression of Phosphatidylinositol 3 Kinase on Human Lung Tumors

This example describes the use of CHR3 probes and PIK3CA probes for ISHassays. The example also describes the use of said probes for copynumber enumeration. One skilled in the art will appreciate that methodsthat deviate from these specific methods can also be used tosuccessfully detect a target nucleic acid and/or chromosome.

Materials and Methods Specimens

Individual lung tissue samples obtained from a tissue specimen archivemaintained at Ventana Medical Systems, Inc. (Tucson, Ariz.) were usedfor developing and optimizing PIK3CA dual ISH (DISH), mRNA ISH, andp110α IHC assays. These samples were redundant clinical specimens thathad been de-identified and unlinked from patient information. A tissuemicroarray slide containing 102 lung tissues was obtained from PantomicsInc. (Richmond, Calif., USA). Paraffin sections (4 μm) containing tissuecores of formalin-fixed, paraffin-embedded breast tissue were mounted onSuperfrost® Plus glass slides.

PIK3CA and Chromosome 3 Oligo Probes

The single-stranded oligonucleotide PIK3CA probe (PIK3CA oligo probe) isa DNP-labeled, repeat-free genomic probe that specifically targets thePIK3CA gene region. The PIK3CA oligo probe spans 759,736 nucleotides(nt) (178,640,071-179,399,807) of genomic DNA from human Chromosome 3,encompassing the PIK3CA target region (UCSC Genome Browser onGRCh37/hg19 human genome Assembly) (FIG. 1). A bioinformatic search wasused to identify PIK3CA specific nucleic acid sequences around thePIK3CA target region. The selected genomic target nucleic acid sequencewas separated into consecutive non-overlapping 80 nt segments. Onethousand two hundred and twelve (1212) ˜80mer oligonucleotides eachcarrying 5 DNP haptens were synthesized. The oligonucleotides werepurified and verified with Mass Spec and gel electrophoresis. Thesingle-stranded oligonucleotide Chr3 probe (Chr3 oligo probe) is a poolof 18 oligonucleotides with a length as specified in Table 4. Eacholigonucleotide was labeled with 2 DIG haptens; the oligonucleotideswere PAGE purified and their molecular weights were verified by massspectrometer. The PIK3CA oligo probe (5.0 ug/ml) and the Chr3 oligoprobe (0.75 ug/ml) were combined in a formamide-containing bufferwithout human blocking DNA.

Automated Bright-Field PIK3CA/CHR3 Oligo Dual ISH for Interphase Slides

The BenchMark ULTRA automated slide processing system (Ventana MedicalSystems, Inc., Tucson, Ariz., USA) was used for designing and evaluatingthe performance of the single-stranded oligonucleotide PIK3CA and CHR3dual ISH assays (PIK3CA/CHR3 Oligo DISH) for PIK3CA and CHR3 DNAtargets. The ultraView SISH and ultraView Alkaline Phosphatase Red ISHdetection kits (Ventana) were used for silver (PIK3CA) and red (CHR3)detection. The slides were deparaffinized at 69° C., followed byincubation with pH 6 citrate buffer at 82° C. and by digestion with ISHProtease 3 for 20 minutes. The probe mixture was then deposited onto theslide so as to contact the sample. The probe(s) were heated to 80° C.for 8 minutes to ensure the probes were denatured. Subsequently,conditions suitable for hybridizing the probes were maintained for 1hour (e.g. temperature was lowered to 44° C. and maintained). Theunbound probes were washed from the sample using 3 stringency washes (pH6.0 citrate buffer at 72° C.). The presence of the probes on the samplewas then detected by contacting the sample with a horseradishperoxidase-labeled rabbit anti-DNP antibody. Following a rinsing step toremove the unbound antibody-enzyme conjugate, the ultraView SISH reagentwas contacted with the sample to cause silver metal precipitate to form.The specific hybridization of the DNP-linked PIK3CA probe to its targetwas detected by visualizing the insoluble precipitate of silverchromogen. The ultraView Alakaline Phosphatase reagents were thencontacted with the sample according to its normal usage, firstcontacting the sample with an alkaline phosphatase (AP)-labeled mouseanti-DIG antibody, followed by rinsing and contacting the sample withthe Fast Red chromogen system. The Chromosome 3 probe was detected byvisualizing the soluble precipitate of the AP-based Fast Red chromogenicsystem. The slides were counterstained by contacting the samples withhematoxylin for 4 minutes and post-counterstained with bluing reagentfor 4 minutes so that the morphology of the tissue could be bettervisualized.

Automated Bright-Field PIK3CA/CHR3 Oligo DISH Chromosome MetaphaseSpread Staining:

Metaphase chromosomes (CGH Metaphase Target Slides, Abbott Molecular)were UV cross-linked on Stratalinker 2400 (Stratagene Model # C00518) atenergy level 200 mJ. They were then treated with 1% trypsin (Sigmacat#T1426) at room temperature for 5 seconds. The slides were thenprocessed for PIK3CA/CHR3 Oligo DISH staining under the same conditionsas described above except that the steps for baking, deparaffin, cellconditioning and counterstaining were omitted. After ISH staining wascompleted on the instrument, slides were stained with 4% Giemsa (Gibco,cat#10092-03) diluted in Gurr buffer (Gibco, cat#10582-013) at roomtemperature for 5 min, and staining was visualized with a regular lightmicroscope.

Bright-Field PIK3CA mRNA ISH

Homo sapiens phosphatidylinositol-4,5-bisphosphate 3-kinase, catalyticsubunit alpha (PIK3CA), mRNA (GenBank accession number: NM_006218.2) wastargeted with Hs-PIK3CA (Cat. No: 603041, Advanced Cell Diagnostics,(ACD), Hayward, Calif.). Thirty of double Z oligonucleotide probe pairswere designed to cover the region 420-3261 nt. The signal amplificationsystem consists of the preamplifier, amplifier, and enzyme conjugatedlabel probe, which assemble into a tree-like structure via sequentialhybridization. To ensure specificity, only when both ZZ probes bind tothe preamplifier binding site can the signal amplification occurs at thetarget site under given assay conditions. Hs-PPIB was used as positivecontrol probe to target Peptidylprolyl Isomerase B (Cyclophilin B) (ACDCat. 313901); and dapB was used as negative control probe to targetbacteria B. subtilis gene dihydrodipicolinate reductase (ACD Cat.310043). The RNA in situ hybridization was performed manually usingRNAscope® 2.0 FFPE Reagent Kit (RNAscope® 2.0 HD Red Reagent Kit, ACDCat. No 310036) according to the manufacturer's instructions. Briefly,FFPE tissue sections were pretreated with heat and protease prior tohybridization with the target oligo probes. Preamplifier, amplifier andAP-labeled oligos were then hybridized sequentially, followed bychromogenic precipitate development. Each sample was quality controlledfor RNA integrity with the probe specific to PPIB RNA and for backgroundwith the probe specific to bacterial dapB RNA. Samples with >70% ofcells stained with moderate and strong signals for PPIB were consideredpass for RNA integrity. Specific RNA staining signal was identified asred, punctuate dots mainly in the cytoplasm. Samples were counterstainedwith Gill's Hematoxylin. Signals were granular and discrete red signalscorresponding to individual PIK3CA mRNA targets. Stained slides werescored semi-quantitatively using conventional bright-field microscopy

Immunohistochemistry of p110α Protein

PI3Kinase p110α (C73F8) rabbit monoclonal antibody (Cell signaling Cat#4249) was diluted 1:50 in 50 mM Tris buffer (pH 7.7) with 1% BSA and 1%normal goat serum. Staining was done on automated BenchMark®Ultrastainers. Antigen recovery was conducted using heat retrieval and CC1standard, a high pH Tris/borate/EDTA buffer (Ventana, Cat#950-224). ThePI3Kinase p110α antibody was incubated at 37° C. for 8 min. The primaryantibody was detected using OptiView DAB IHC detection kit (Ventana,Cat#760-700). Slides were counter-stained with hematoxylin II (Ventana,Cat#790-2208) for 8 min, followed by Bluing reagent (Ventana,Cat#760-2037) for 4 min.

PIK3CA/CHR3 Oligo DISH Stain Scoring Criteria:

Three board-certified pathologists (W. C., J. J., and T. G.) that had noprevious knowledge of the genetic, clinical and IHC results evaluatedthe DISH stained slides. The readers first identify an area ofneoplastic nuclei with most copy numbers, then count PIK3CA and CHR3signals in 50-100 neoplastic nuclei and 50 adjacent mesenchymal nuclei(or normal epithelial nuclei) if possible. If one area does not haveenough neoplastic nuclei to count (e.g. focal amplification),pathologists may move to another area with most copy numbers.

Bright-Field PIK3CA mRNA ISH Stain Semi-Quantitative Scoring Criteria

Scoring performed at 20× magnification. The scoring system is asfollowing: 0, no staining or <1 dot/10 cells; 1, 1-3 dots/cell; 2, 4-9dots/cell, none or very few dot clusters; 3. 10-15 dots/cell and <10%dots are in clusters; 4, >15 dots/cell and >10% dots are in clusters. If<5% of cells score 1 and >95% of cells score 0, a score of 0 will begiven. If 5-30% of cells score 1 and >70% of cells score 0, a score of0.5 will be given. ACD's H-Score analysis was also performed to evaluateheterogeneity in PIK3CA expression. The RNA signal is binned into 5groups (bin 0: 0 dots/cell, bin 1: 1-3 dots/cell, bin 2: 4-9 dots/cell,bin 3: 10-15 dots/cell, and bin 4: >15 dots/cell with >10% of dots inclusters). Each sample is evaluated for the percentage of cells in eachbin. The H-Score is calculated by adding up the percentage of cells ineach bin, with a weight assigned to each bin, according to the formulabelow. H-Scores are given on a scale of 0-400. H scores=0*(% of cells inbin 0)+1*(% of cells in bin 1)+2*(% of cells in bin 2)+3*(% of cells inbin 3)+4*(% of cells in bin 4)

p110α IHC Stain Scoring Criteria

By light microscopy, representative viable tissue sections were scoredsemiquantitatively for cytoplasmic staining. The dominant stainingintensity in tumor cells was scored as: 0=negative;

1=weak; 2=intermediate; 3=strong. The level of staining in adjacentmesenchymal cells was used as baseline references. Most p110α IHC stainstains were homogeneous with more than 30% of cells showing the dominantstaining intensity.

Results:

PIK3CA/CHR3 Oligo DISH Assay with 1 hr Hybridization and No Use of HumanBlocking DNA: Specific, Robust, and Superior CHR3 Staining

Specificity of the PIK3CA/CHR3 Oligo DISH assay was first verified onchromosomal metaphase spreads slides without human blocking DNA. ThePIK3CA oligo probe (black signal) and the CHR3 oligo probe (red signal)were localized to the same chromosome. No cross-hybridization of eitherthe PIK3CA oligo probe or the CHR3 oligo probe to other chromosomes wasobserved (FIG. 2). The specificity of PIK3CA/CHR3 Oligo DISH assay wasfurther assessed on 5 non-tumor lung tissues (3 normal lung and 2tuberculosis, TB granuloma cases). The average PIK3CA copy number ranges1.69 to 1.86 per nuclei, the average CHR3 copy number ranges 1.75 to1.97 per nuclei, and the average PIK3CA/CHR3 ratio ranges 1.701 to 1.08per nuclei (Table 6). Furthermore, PIK3CA/CHR3 Oligo DISH assay wastested on Calu 3 cell line xenograft which has been characterized PIK3CAamplification (Spoerke, O'Brien et al. 2012) (FIG. 3).

TABLE 6 PIK3CA AND CHROMOSOME 3 COPY NUMBERS IN NORMAL AND BENIGN LUNGTISSUES Ave Ave Nuclei PIK3CA Chr3 PIK3CA/CHR3 PIK3CA CHR3 Cases TypeNumber copies copies Ratio copies copies Case 1 Normal 100 182 197 1.021.82 1.97 Case 2 Normal 100 186 192 1.01 1.86 1.92 Case 3 Normal 100 175172 1.08 1.75 1.72 Case 4 Tuberculosis, 100 173 176 1.05 1.73 1.76 TBgranuloma Case 5 Tuberculosis, 100 169 175 1.03 1.69 1.75 TB granuloma

All the aforementioned staining was performed with 1 hr hybridization, atime course study was designed to test if 1 hr is sufficient to generateadequate staining results when comparing to the longer hybridizationtimes (2, 3, 4, 5, and 6 hrs). All the time points including 1 hrgenerate PIK3CA and CHR3 staining intensity 2.5 and above (≧2acceptable), and staining coverage 80% and above (≧50% acceptable), andless than 0.5 background (≦1 acceptable) (FIG. 4).

Most FISH studies targeted chromosome 3 centromere alpha satellite(D3Z1) sequence for CHR3 enumeration with plasmid pHS05 (EMBL accessionnumber Z12006) (Alexandrov, Mitkevich et al. 1988, Alexandrov, Mashkovaet al. 1993). These assays all require human blocking DNA to reducebackground from cross-reactivity to other chromosome alpha satellite. Welabeled plasmid pHS05 with DIG and compared its performance to that ofthe CHR3 Oligo Probe. First of all, in the absence of human blockingDNA, the plasmid pHS05 probe generated excessive number of red signalsin the epithelial and stromal nuclei in a normal lung tissue (FIG. 5).Next, we bulked the plasmid pHS05 probe with human placenta blocking DNA(2 mg/ml) and stained with the CHR3 Oligo Probe pair-wise on the samelung specimens. Whereas the chromosome 3 signals stained with theplasmid pHS05 probe appeared to be specific (1-2 dots/nuclei), thesignals often looked diffuse or smudge-like (FIGS. 6A&B). Moreover, insevere scenario, enumeration of discrete dots was a big challenge. Incontrast, the CHR3 Oligo Probe usually generated discrete signals withregular size and shape (FIGS. 6C&D).

Scoring 50 Neoplastic Nuclei with Most Copy Numbers for the PIK3CA/CHR3Oligo DISH Assay

Up to now, no scoring criterion is available for bright-field PIK3CA/CHRdual ISH assay on lung tissues. FISH studies usually counted ˜100 nucleion average. The minimal number is 20 on 419 primary tumor samples(Jehan, Bavi et al. 2009) (Kiyose, Nagura et al. 2012) counted 30 to 50nuclei, and Costa C. et al. (Costa, Espinet et al. 2009) counted 200 in26 primary tumor samples. PIK3CA and CHR3 signals were enumerated on 100nuclei on 5 non-neoplastic lung tissues, 15 SCC tissues, and 6 otherlung tumor types. PIK3CA/CHR3 ratio from the highest scores of 50 nucleiwas consistent with the scores from the entire 100 nuclei enumeration(FIG. 7). When sequentially selecting 50 nuclei, 25 out of 26 casesdemonstrated consistent results (either ratio <2 as normal or >2 asamplified) except for one case. This case had a ratio >2 with 100 nucleicounts, but <2 with 50 nuclei that were sequentially selected (FIG. 7).These results prove 50 nuclei count is sufficient enough to generateconsistent results as 100 nuclei; and also underscore the importance ofcounting neoplastic nuclei with most copy numbers (See MATERIALS ANDMETHODS “PIK3CA/CHR3 Oligo DISH Stain Scoring Criteria”). Counting 50neoplastic nuclei with most copy numbers was therefore implemented forthe rest of clinical specimen assessment.

PIK3CA Gene Amplification Mainly Occurs in SCC (37%) by the PIK3CA/CHR3Oligo DISH Assay

PIK3CA gene status was analyzed on a cohort of 102 lung tissuemicroarray specimens: 49 SCC, 26 adenocarcinomas, 11 bronchialcarcinomas, 1 carcinoid, 1 clear cell carcinomas, 3 normal, 1 papillaryadenocarcinoma, 3 small cell lung carcinomas (SCLC), 2 tuberculosis, TBgranulomas, and 5 undifferentiated carcinomas. 100% 1st pass rate wasachieved on the 102 tissues, in which all the staining is interpretablefor PIK3CA and CHR3 copy numbers. The PIK3CA/CHR3 ratio, average PIK3CAcopy per nuclei, and average CHR3 copy per nuclei for each of the 102lung tissues are illustrated in FIG. 8. In this cohort, SCC stands outwith the highest incidence of elevated ratio and PIK3CA copy number fromother tumor types. Only 1 adenocarcinoma with 4 copies of CHR3 and 1 SCCwith 3.16 copies of CHR3 (FIG. 8), chromosome 3 polysomy seems unlikelythe driving force for PIK3CA copy number gain.

A total of 21 lung tissues have PIK3CA/CHR3 ratio greater than 2 and/oraverage PIK3CA copy number greater than 4, among which 15 have bothratio >2 and copy number >4. These include 18 SCC (36.7% of SCC), 2adenocarcinomas (7.7% of adenocarcinomas) and 1 SCLC (1 out of 3 SCLC)(FIG. 9). The PIK3CA/CHR3 ratio ranges from 2.03 to 5.77; the averagePIK3 copy number per nuclei ranges from 3.00 to 9.96; the average CHR3copy number per nuclei ranges from 1.38 to 4.02. Majority (18 out of 21)cases with PIK3CA copy number gain manifest multiple copies of discretesignals. Among the 21 cases, 20 demonstrate focal PIK3CA amplificationin chromosome 3, only 1 case (C10, adenocarcinoma) has chromosome 3polysomy.

Heterogeneity of PIK3CA gene copy gain in the 21 cases is presented inTable 7. In Case C12, C13, E6, F1, F2, F7, F8, and F10, almost allnuclei carry abnormally high copy number (4-9 and above per nuclei). Inthe remaining 13 cases, 12-80% of nuclei with normal (0-3) PIK3CA copiesare mixed with abnormal (4-9 and above) nuclei. Regarding the copynumber ranges of 4-9/nuclei, >10/nuclei, and >15/nuclei, 4-9 copies pernuclei represents the largest population of nuclei with PIK3CA geneamplification.

TABLE 7 HETEROGENEITY OF PIK3CA GENE COPY NUMBER GAIN IN THE 21 LUNGTUMORS Tumor % 0-3 % 4-9 % >10 % >15 Cores type #Nuclei copies copiescopies copies A11 SCLC 100 41 55 4 B12 AdenoCA 50 40 60 C10 AdenoCA 5038 60 2 C12 SCC 50 68 28 4 C13 SCC 50 2 76 14 8 D4 SCC 100 33 62 5 D6SCC 100 66 34 E3 SCC 50 42 58 E6 SCC 50 2 68 24 6 E8 SCC 50 38 62 E9 SCC50 42 56 2 E10 SCC 50 22 56 20 2 E13 SCC 50 12 74 14 F1 SCC 50 2 92 4 F2SCC 50 0 50 42 8 F6 SCC 50 10 76 14 F7 SCC 50 0 58 36 6 F8 SCC 50 0 5232 14 F10 SCC 50 2 82 8 8 F11 SCC 50 26 72 2 G8 SCC 50 80 20

With these 102 lung tissues, we also evaluated the CHR3 stainingmorphology with pHS05 plasmid in comparison to the CHR3 Oligo Probe.Thirty-seven (37) tissues were commented for inferior CHR3 stainingquality (e.g. more red smudges, more red smears, increased redbackground), while only 2 cases stained with CHR3 Oligo Probe werecommented for red signal variability.

PIK3CA mRNA Upregulation and Protein Overexpression Mainly Occurs in SCCby the PIK3CA mRNA ISH (45.3%) and p110 αIHC (75.5%)

The PIK3CA mRNA ISH staining achieved 87.3% (89/102) pass rate. Thirteen(13) tissues failed QC as <70% of cells with 2+ intensity for PPIBstaining. Thirty (30) cases have mRNA H score 70 and above, among which24 are SCC (24/49, 49.0% of SCC), 3 adenocarcinoma (3/26, 11.5%), 1 SCLCand 2 Undifferentiated. Non-tumor tissues have the lowest mRNAexpression (3 normal and 2 TB) (FIG. 10C).

To optimize p110α IHC assay condition, human kidney was selected for itsknown IHC staining patterns (Uhlen M 2010), and Calu-3 cell line waschosen for its known PIK3CA gene amplification. Cells in glomerulishowed low-to-medium staining (<25% of cells with strong intensity),while cells in tubules had high staining (>75% of cells with strongintensity). Calu-3 had high immunoreactive staining. p110α IHC stainingfeatured a predominantly cytoplasmic and in some cases an accentuatedmembranous staining (FIG. 10A). Focal and weak positive staining wasfound in some bronchiolar epithelial cells of normal lung tissue, andsome of the lymphocytes and macrophages of tumor area, likely due to therole of PI3K in normal cell proliferation processes.

Ninety-nine (99) cases staining with p110α IHC demonstrated certainlevel of staining (0.5-3), 3 cases exhibited no staining in the entiretissue area. 65 cases exhibited p110α IHC intensity 2+ (34 cases) and 3+(31 cases), among which 40 were SCC (40/49, 81.6% of SCC), 14 wereadenocarcinomas (14/26, 53.8%), and 11 for other tumor types. Non-tumortissues (3 normal and 2 TB) had the lowest p110α IHC intensity (0.5-1)(FIG. 10B).

17 Out of the 21 Amplified Cases (80.9%) have Both mRNA and ProteinOverexpression (15 Cases) and Protein Overexpression (2 Cases)

For the 21 cases with PIK3CA copy number gain, fifteen cases (A11, C12,C13, D4, D6, E3, E6, E8, E10, E13, F2, F8, F10, F11, and G8) have bothelevated mRNA level (H score >70) and protein overexpression (intensity2 and 3). Two cases (C10 and F1) have p110alpha overexpression(intensity 2 and 3), but mRNA H scores are below 70 (22 and 44,respectively). Three cases (B12, E9 and F7) have no over-expression atboth mRNA and protein levels. One case (F6) has a low H score (5) formRNA, and the IHC staining is not interpretable. Taken together, ˜80% ofthe cases with PIK3CA copy number gain have overexpression at mRNAand/or protein levels. FIG. 10A showed the images of PIK3CA/CHR3 DISH,PIK3CA mRNA ISH and p100α IHC staining on a SCC (F3) with normal genecopy numbers (2.16, ratio 0.98), borderline mRNA expression (H score 75)and normal p110α expression (intensity 1, 80%). FIG. 10B showed theDISH, mRNA ISH and IHC images on a SCC (F2) with gene copy gain (9.04,ratio 3.55), increased mRNA (H score 160) and protein expression(intensity 3, 80%) (FIGS. 11A, 11B and 11C).

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety for allpurposes. In case of conflict, the present specification, includingexplanations of terms, will control. The following other patents areherein incorporated by reference in their entirety: U.S. Pat. No.7,807,356; U.S. Pat. No. 8,445,206.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof as being present inthe disclosure. It is therefore intended that the following appendedclaims and claims hereafter introduced are interpreted to include allsuch modifications, permutations, additions and sub-combinations as arewithin their true spirit and scope.

Additional Exemplary Embodiments

The following additional embodiments are also specifically disclosed.This is not an exhaustive list.

-   1. A system for in situ hybridization comprising:    -   a control probe specific to a control region of chromosome 3,        wherein the control probe is labeled with at least one first        label, and wherein the control probe is configured to achieve        least two enumerable signals per cell with a staining intensity        of ≧2 and staining coverage of ≧50% of the number of total        nuclei of a control sample within 3 hours of hybridization;    -   wherein each enumerable signal has a generally round shape,    -   wherein a round shape is a simple closed curve that fits within        a first region, the first region lies on and outside of an inner        circle and on and inside of a concentric outer circle, the inner        circle having an inner radius (R_(in)) and the outer circle        having an outer radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out),    -   wherein a “simple closed curve” as used herein is a connected        curve that does not cross itself and ends at the same point        where it begins.-   2. The system of embodiment 1, wherein the control probe is a first    plurality of single-stranded oligonucleotide probes, each probe    comprising:    -   a sequence selected from the group consisting of SEQ ID NOs:        1-18; or    -   a sequence selected from the group consisting of a truncated        version of SEQ ID NOs: 1-18, the truncated version being at        least 40 contiguous basepairs (bp) of said SEQ ID NOs: 1-18; or    -   a sequence selected from the group consisting of a sequence that        has at least 70% sequence identity to one of SEQ ID NOs: 1-18.-   3. The system of embodiment 1, wherein the control probe is a first    plurality of single-stranded oligonucleotide probes, each probe    comprising:    -   a sequence selected from the group consisting of SEQ ID NOs:        1-18; or    -   a sequence selected from the group consisting of a truncated        version of SEQ ID NOs: 1-18, the truncated version being at        least 40 contiguous bp of said SEQ ID NOs:1-18; or    -   a sequence selected from the group consisting of a sequence that        has at least 80% sequence identity to one of SEQ ID NOs: 1-18.-   4. The system of embodiment 1, wherein the control probe is a first    plurality of single-stranded oligonucleotide probes, each probe    comprising:    -   a sequence selected from the group consisting of SEQ ID NOs:        1-18; or    -   a sequence selected from the group consisting of a truncated        version of SEQ ID NOs: 1-18, the truncated version being at        least 40 contiguous bp of said SEQ ID NOs:1-18; or    -   a sequence selected from the group consisting of a sequence that        has at least 90% sequence identity to one of SEQ ID NOs: 1-18.-   5. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes is configured to    hybridize uniquely and specifically to a portion of the control    region human chromosome 3 so that other chromosomes or portions    thereof are not evidently labeled.-   6. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes is configured to    hybridize uniquely and specifically to a portion the control region    human chromosome 3 so that other chromosomes or portions thereof are    not evidently labeled without the influence of blocking DNA.-   7. The system of any of embodiments 2 to 6, wherein the control    probes each comprise between 50 to 100 nucleotides.-   8. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes target between 2    and 18 distinct portions within the control region.-   9. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes target between 4    and 18 distinct portions within the control region.-   10. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes target between 6    and 18 distinct portions within the control region.-   11. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes target between 8    and 18 distinct portions within the control region.-   12. The system of any of embodiments 2 to 4, wherein the first    plurality of single-stranded oligonucleotide probes target between    10 and 18 distinct portions within the control region.-   13. The system of any of embodiments 2 to 12, wherein the control    probes are each labeled with at least 2, at least 3, at least 4, or    at least 5 first labels.-   14. The system of any of embodiments 1 to 13, wherein the first    label comprises a digoxigenin (DIG).-   15. The system of any of embodiments 1 to 14, further comprising a    target probe specific to a target region of human chromosome 3,    wherein the target probe is labeled with at least one second label.-   16. The system of embodiment 10, wherein the target probe is    specific to a target region near or around the PIK3CA gene locus.-   17. The system of embodiment 10, wherein the target probe is    specific to a region between nucleotides 178,640,071 and 179,399,807    of human chromosome 3.-   18. The system of any of embodiments 1 to 17, further comprising an    ISH staining instrument, the instrument is configured to contact the    control probe to a tissue sample.-   19. A kit comprising a vessel containing a system according to any    of embodiments 1 to 18 and instructions for use.-   20. A slide comprising a plurality of nuclei chromogenically stained    for a chromosome, wherein more than 50% of the nuclei have    enumerable signals for said chromosome, each enumerable signal being    a generally round shape,    -   wherein a round shape is a simple closed curve that fits within        a first region, wherein the first region lies on and outside of        an inner circle and on and inside of a concentric outer circle,        the inner circle having an inner radius (R_(in)) and the outer        circle having an outer radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out),    -   wherein a “simple closed curve” as used herein is a connected        curve that does not cross itself and ends at the same point        where it begins.-   21. The slide of embodiment 20, wherein the chromosome is human    chromosome 3.-   22. The slide of embodiment 20 or 21, wherein the slide is made    using a system according to any of the claims 1 to 18.-   23. The slide of any of embodiments 20 to 22, wherein more than 60%    of the nuclei have enumerable chromosome signals.-   24. The slide of any of embodiments 20 to 22, wherein more than 70%    of the nuclei have enumerable chromosome signals.-   25. The slide of any of embodiments 20 to 24, wherein the inner    radius is no less than 60% of the outer radius.-   26. The slide of any of embodiments 20 to 24, wherein the inner    radius is no less than 75% of the outer radius.-   27. The slide of any of embodiments 20 to 24, wherein the inner    radius is no less than 90% of the outer radius.-   28. The slide of any of embodiments 20 to 24, wherein the outer    radius is between about 0.25 to 0.675 μm.-   29. The slide of any of embodiments 20 to 24, wherein the outer    radius is between about 0.2 to 0.75 μm.-   30. The slide of any of embodiments 20 to 24, wherein the outer    radius is between about 0.15 to 1 μm.-   31. The slide of any of embodiments 20 to 24, wherein the average    outer radius of the enumerable signals is between about 0.2 to 0.75    μm.-   32. The slide of any of embodiments 20 to 24, wherein the average    outer radius of the enumerable signals has a standard deviation of    less than 0.5 μm.-   33. The slide of any of embodiments 20 to 24, wherein the average    outer radius of the enumerable signals has a standard deviation of    less than 0.25 μm.-   34. A slide comprising a plurality of nuclei stained for a    chromosome,    -   wherein a sum of the surface area covered by a staining signal        is assigned a 100% value,    -   wherein at least 50% of said sum of the surface area is derived        from discrete enumerable, mono-sized round signals, each        enumerable round signal being a generally round shape,    -   wherein a round shape is a simple closed curve that fits within        a first region, wherein the first region lies on and outside of        an inner circle and on and inside of a concentric outer circle,        the inner circle having an inner radius (R_(in)) and the outer        circle having an outer radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out).-   35. The slide of embodiment 34, wherein the chromosome is human    chromosome 3.-   36. The slide of embodiments 34 or 35, wherein the slide is made    using a system according to any of the claims 1 to 18.-   37. The slide of any of embodiments 34 to 36, wherein more than 60%    of said sum of the surface area is from discrete mono-sized round    signals; wherein mono-sized round signals have a round shape,    wherein a round shape is a simple closed curve that fits within a    first region, wherein the first region lies on and outside an inner    circle and on and inside a concentric outer circle, the inner circle    having an inner radius (R_(in)) and the outer circle having an outer    radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out).-   38. The slide of any of embodiments 34 to 38, wherein more than 70%    of said sum of the surface area is from discrete mono-sized round    signals; wherein mono-sized round signals have a round shape,    wherein a round shape is a simple closed curve that fits within a    first region, wherein the first region lies on and outside an inner    circle and on and inside a concentric outer circle, the inner circle    having an inner radius (R_(in)) and the outer circle having an outer    radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out).-   39. The slide of any of embodiments 34 to 38, wherein the inner    radius is no less than 60% of the outer radius.-   40. The slide of any of embodiments 34 to 38, wherein the inner    radius is no less than 75% of the outer radius.-   41. The slide of any of embodiments 34 to 38, wherein the inner    radius is no less than 90% of the outer radius.-   42. A method for in situ hybridization comprising:    -   contacting a tissue sample with a control probe specific to a        control region of a chromosome, wherein the control probe is a        single-stranded oligonucleotide probe labeled with at least one        first label, and wherein the control probe is configured to        achieve at least two signals per cell with a staining intensity        of ≧2 and staining coverage of ≧50% of the number of total        nuclei of a control sample within 3 hours of hybridization;    -   hybridizing the control probe to the control region under        conditions for a period of time less than about 3 hours;    -   rinsing the sample to remove unbound probe; and    -   detecting the presence of the hybridized probe.-   43. The method of embodiment 42, wherein the method is for    bright-field in situ hybridization.-   44. The method of embodiment 42 or 43, wherein the control probe is    hybridized to the control region under conditions for a period of    time less than about 2 hours.-   45. The method of embodiment 42 or 43, wherein the control probe is    hybridized to the control region under conditions for a period of    time less than about 1 hour.-   46. The method of any of embodiments 42 to 45, wherein the    chromosome is human chromosome 3.-   47. The method of any of embodiments 42 to 46, further comprising    contacting the tissue sample with a target probe specific to a    control region of the chromosome, the target probe is a    single-stranded oligonucleotide probe labeled with at least one    second label.-   48. The method of embodiment 47, wherein the target probe is    specific to a target region near or around the PIK3CA gene locus of    chromosome 3.-   49. The method of embodiment 47, wherein the target probe is    specific to a region between nucleotides 178,640,071 and 179,399,807    of human chromosome 3.-   50. The method of any of embodiments 42 to 49, further comprising    applying chromogenic detection reagents that recognize the first    label and amplify the signal associated with said first label.-   51. The method of any of embodiments 42 to 50, wherein the tissue    sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.-   52. The method of any of embodiments 42 to 51, wherein the method is    free from the use of blocking DNA.-   53. The method of any of embodiments 42 to 51, wherein an amount of    blocking DNA is used.-   54. A method for in situ hybridization of a tissue sample, the    method comprising contacting the tissue sample with a system    according to any of claims 1 to 18.-   55. A method for bright-field chromogenic in situ hybridization    without the use of blocking DNA, said method comprising:    -   contacting a tissue sample with a control probe specific to a        control region of a chromosome;    -   hybridizing the control probe to the control region of said        chromosome;    -   rinsing the sample to remove unbound probe; and    -   detecting the presence of the hybridized probe via a chromogenic        reagent;    -   wherein no blocking DNA is used in any of the above steps.-   56. A method for obtaining two bright-field chromogenic in situ    hybridization signals per cell, said method comprising:    -   contacting a tissue sample containing a plurality of cells with        a control probe specific to a control region of a single        chromosome, wherein the probe selected so as to not evidently        bind non-specifically in the absence of blocking DNA;    -   hybridizing the control probe to the control region of said        chromosome;    -   rinsing the sample to remove unbound probe; and    -   detecting the presence of the hybridized probe via a chromogenic        reagent so as to generate two bright-field chromogenic in situ        hybridization signals per cell.-   57. The method of embodiment 55 or 56, wherein the control probe    comprises a single-stranded oligonucleotide probe labeled with at    least one first label.-   58. The method of any of embodiments 55 to 57, wherein the    chromosome is human chromosome 3.-   59. A method for bright-field chromogenic in situ hybridization    comprising:    -   contacting a tissue sample with a control probe specific to a        control region of a chromosome;    -   hybridizing the control probe to the control region of said        chromosome;    -   rinsing the sample to remove unbound probe; and    -   detecting the presence of the hybridized probe via a chromogenic        reagent;    -   wherein an amount of blocking DNA is used in one of the above        steps, the amount of blocking DNA is sufficient to block out no        more than 50% of the non-specific binding.-   60. The method of embodiment 59, wherein the control probe comprises    a single stranded oligonucleotide probe labeled with at least one    first label.-   61. The method of embodiment 59 or 60, wherein the chromosome is    human chromosome 3.-   62. The method of any of embodiments 59 to 61, wherein the amount of    blocking DNA is between about 1 pg/ml to 1 mg/ml.-   63. A method of in situ hybridization, the method comprising:    -   contacting a tissue sample with a control probe specific to a        control region of a chromosome, wherein the control probe is a        single-stranded oligonucleotide probe labeled with at least one        first label, and wherein the control probe is configured to        achieve two signals per cell with a staining intensity of ≧2 and        staining coverage of ≧50% of the number of total nuclei of a        control sample within 3 hours of hybridization;    -   hybridizing the control probe to the control region of said        chromosome under conditions for a period of time less than 3        hours;    -   rinsing the sample to remove unbound probe; and    -   detecting the presence of the hybridized probe;    -   wherein more than 50% of the nuclei of the tissue sample have        enumerable signals for said chromosome, an enumerable signal        being a generally round shape, wherein a round shape is a simple        closed curve that fits within a first region, wherein the first        region lies on and outside an inner circle and on and inside a        concentric outer circle, the inner circle having an inner radius        (R_(in)) and the outer circle having an outer radius (R_(out)),    -   wherein the simple close curve has a radius R_(simple),    -   wherein R_(in)≦R_(simple)≦R_(out), and    -   wherein, R_(in) is ≧50% of R_(out).-   64. The method of embodiment 63, wherein background signals (more    than 2 signals) are not observed in >80% of cells of the tissue    sample.-   65. The method of embodiment 63 or 64, wherein background signals    have a staining intensity of 0 or 1.-   66. The method of any of embodiments 63 to 65, wherein the tissue    sample is contacted with a system according to any of the claims 1    to 19.-   67. The method of any of embodiments 63 to 66, wherein more than 60%    of the nuclei have enumerable chromosome signals.-   68. The method of any of embodiments 63 to 66, wherein more than 70%    of the nuclei have enumerable chromosome signals.-   69. The method of any of embodiments 63 to 68, wherein the inner    radius is no less than 60% of the outer radius.-   70. The method of any of embodiments 63 to 68, wherein the inner    radius is no less than 75% of the outer radius.-   71. The method of any of embodiments 63 to 68, wherein the inner    radius is no less than 90% of the outer radius.-   72. The method of any of embodiments 63 to 71, wherein the outer    radius is between about 0.2 to 0.675 μm.-   73. The method of any of embodiments 63 to 71, wherein the outer    radius is between about 0.2 to 0.75 μm.-   74. The method of any of embodiments 63 to 71, wherein the outer    radius is between about 0.1 to 0.5 μm.-   75. The method of any of embodiments 63 to 71, wherein the average    outer radius of the enumerable signals is between about 0.2 to 0.75    μm.-   76. The method of any of embodiments 63 to 71, wherein the average    outer radius of the enumerable signals has a standard deviation of    less than 0.5 μm.-   77. The method of any of embodiments 63 to 71, wherein the average    outer radius of the enumerable signals has a standard deviation of    less than 0.2 μm.-   78. The method of any of embodiments 63 to 77, wherein the    chromosome is human chromosome 3.-   79. A method of scoring for a chromosome for PIK3CA gene copy    amplification, said method comprising:    -   obtaining a tissue sample having undergone ISH according to        claims 42-78, wherein a control probe and target probe are used;    -   identifying an area of neoplastic nuclei with most copy numbers;        and    -   counting enumerable signals for PIK3CA signal in 50-100        neoplastic nuclei and either 50 adjacent mesenchymal nuclei or        50 adjacent normal epithelial nuclei;    -   wherein scoring criteria comprises: no staining or <1 dot/10        cells is scored as 0; 1-3 dots/cell is scored as 1; 4-9        dots/cell, none or very few dot clusters is scored as 2; 10-15        dots/cell and <10% dots are in clusters is scored as 3; and >15        dots/cell and >10% dots are in clusters is scored as 4.-   80. The method of embodiment 79 further comprising calculating the    ratio of PIK3CA signal to control signal.-   81. The method of embodiment 79 further comprising calculating the    average number of PIK3CA copies per nuclei.-   82. A control probe for use in a bright-field chromogenic in situ    hybridization, wherein the control probe is specific to a control    region of chromosome 3, the control probe is labeled with at least    one first label, the control probe is selected so as to not    evidently bind non-specifically in the absence of blocking DNA.

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1. A system for in situ hybridization comprising: a control probespecific to a control region of chromosome 3, wherein the control probeis labeled with at least one first label, and wherein the control probeis configured to achieve least two enumerable signals per cell with astaining intensity of ≧2 and staining coverage of ≧50% of the number oftotal nuclei of a control sample within 3 hours of hybridization;wherein each enumerable signal has a generally round shape, wherein around shape is a simple closed curve that fits within a first region,the first region lies on and outside of an inner circle and on andinside of a concentric outer circle, the inner circle having an innerradius (R_(in)) and the outer circle having an outer radius (R_(out)),wherein the simple close curve has a radius R_(simple), whereinR_(in)≦R_(simple)≦R_(out), and wherein, R_(in) is ≧50% of R_(out),wherein a “simple closed curve” as used herein is a connected curve thatdoes not cross itself and ends at the same point where it begins.
 2. Thesystem of claim 1, wherein the control probe is a first plurality ofsingle-stranded oligonucleotide probes, each probe comprising: asequence selected from the group consisting of SEQ ID NOs: 1-18; or asequence selected from the group consisting of a truncated version ofSEQ ID NOs: 1-18, the truncated version being at least 40 contiguousbasepairs (bp) of said SEQ ID NOs:1-18; or a sequence selected from thegroup consisting of a sequence that has at least 70% sequence identityto one of SEQ ID NOs: 1-18.
 3. The system of claim 1, wherein thecontrol probe is a first plurality of single-stranded oligonucleotideprobes, each probe comprising: a sequence selected from the groupconsisting of SEQ ID NOs: 1-18; or a sequence selected from the groupconsisting of a truncated version of SEQ ID NOs: 1-18, the truncatedversion being at least 40 contiguous bp of said SEQ ID NOs:1-18; or asequence selected from the group consisting of a sequence that has atleast 80% sequence identity to one of SEQ ID NOs: 1-18.
 4. The system ofclaim 1, wherein the control probe is a first plurality ofsingle-stranded oligonucleotide probes, each probe comprising: asequence selected from the group consisting of SEQ ID NOs: 1-18; or asequence selected from the group consisting of a truncated version ofSEQ ID NOs: 1-18, the truncated version being at least 40 contiguous bpof said SEQ ID NOs:1-18; or a sequence selected from the groupconsisting of a sequence that has at least 90% sequence identity to oneof SEQ ID NOs: 1-18.
 5. The system of claim 2, wherein the firstplurality of single-stranded oligonucleotide probes is configured tohybridize uniquely and specifically to a portion of the control regionhuman chromosome 3 so that other chromosomes or portions thereof are notevidently labeled.
 6. The system of claim 2, wherein the first pluralityof single-stranded oligonucleotide probes is configured to hybridizeuniquely and specifically to a portion the control region humanchromosome 3 so that other chromosomes or portions thereof are notevidently labeled without the influence of blocking DNA.
 7. The systemof claim 2, wherein the control probes each comprise between 50 to 100nucleotides.
 8. The system of claim 1, further comprising a target probespecific to a target region of human chromosome 3, wherein the targetprobe is labeled with at least one second label.
 9. The system of claim2, wherein the target probe is specific to a target region near oraround the PIK3CA gene locus.
 10. A slide comprising a plurality ofnuclei chromogenically stained for a chromosome, wherein more than 50%of the nuclei have enumerable signals for said chromosome, eachenumerable signal being a generally round shape, wherein a round shapeis a simple closed curve that fits within a first region, wherein thefirst region lies on and outside of an inner circle and on and inside ofa concentric outer circle, the inner circle having an inner radius(R_(in)) and the outer circle having an outer radius (R_(out)), whereinthe simple close curve has a radius R_(simple), whereinR_(in)≦R_(simple)≦R_(out), and wherein, R_(in) is ≧50% of R_(out),wherein a “simple closed curve” as used herein is a connected curve thatdoes not cross itself and ends at the same point where it begins. 11.The slide of claim 10, wherein the chromosome is human chromosome
 3. 12.The slide of claim 10, wherein more than 60% of the nuclei haveenumerable chromosome signals.
 13. The slide claim 10, wherein more than70% of the nuclei have enumerable chromosome signals.
 14. A method forin situ hybridization comprising: contacting a tissue sample with acontrol probe specific to a control region of a chromosome, wherein thecontrol probe is a single-stranded oligonucleotide probe labeled with atleast one first label, and wherein the control probe is configured toachieve at least two signals per cell with a staining intensity of ≧2and staining coverage of ≧50% of the number of total nuclei of a controlsample within 3 hours of hybridization; hybridizing the control probe tothe control region under conditions for a period of time less than about3 hours; rinsing the sample to remove unbound probe; and detecting thepresence of the hybridized probe.
 15. The method of claim 14, whereinthe method is for bright-field in situ hybridization.
 16. The method ofclaim 14, wherein the control probe is hybridized to the control regionunder conditions for a period of time less than about 2 hours.
 17. Themethod of claim 14, wherein the chromosome is human chromosome
 3. 18.The method of claim 14, further comprising contacting the tissue samplewith a target probe specific to a control region of the chromosome, thetarget probe is a single-stranded oligonucleotide probe labeled with atleast one second label.
 19. The method of claim 18, wherein the targetprobe is specific to a target region near or around the PIK3CA genelocus of chromosome
 3. 20. The method of claim 18, wherein the targetprobe is specific to a region between nucleotides 178,640,071 and179,399,807 of human chromosome
 3. 21. The method of claim 14, furthercomprising applying chromogenic detection reagents that recognize thefirst label and amplify the signal associated with said first label. 22.The method of claim 14, wherein the tissue sample is a formalin-fixedparaffin-embedded (FFPE) tissue sample.
 23. A method for in situhybridization of a tissue sample, the method comprising contacting thetissue sample with a system according to any of claim 1.